Name: analyzing the functions of mast cells in vivo using 'mast cell knock-in' mice
Uploaded: 2015-05-27T15:00:00.000+00:00
Duration: 9 min 7 s
Description: Watch this Scientific Journal Video about Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice at JoVE.com

N.G. is the recipient of fellowships from the French &#8220;Fondation pour la Recherche M&#233;dicale FRM&#8221; and the Philipp Foundation; R.S. is supported by the Lucile Packard Foundation for Children&#8217;s Health and the Stanford NIH/NCRR CTSA award number UL1 RR025744; P.S. is supported by a Max Kade Fellowship of the Max Kade Foundation and the Austrian Academy of Sciences and a Schroedinger Fellowship of the Austrian Science Fund (FWF): J3399-B21; S.J.G. acknowledges support from National Institutes of Health grants U19 AI104209, NS 080062 and from Tobacco-Related Disease Research Program at University of California; L.L.R. acknowledges support from the Arthritis National Research Foundation (ANRF) and National Institutes of Health grant K99AI110645.

All animal care and experimentation were conducted in compliance with the guidelines of the National Institutes of Health and with the specific approval of the Institutional Animal Care and Use Committee of Stanford University.
1. Generation and Characterization of Bone Marrow-derived Cultured Mast Cells (BMCMCs).
Note: Donor BMCMCs should be generated from bone marrow cells of the same genetic background as the recipient MC-deficient mice. Male-derived donor BMCMCs are not suitable for engraftment of female mice. Female-derived donor BMCMCs will successfully engraft into both male and female recipients.
<ol>
	<li>Bone Marrow Extraction.
	<ol>
		<li>Pour sterile phosphate-buffered saline (PBS) into 6 well culture plate (1 well per mouse) and place on ice.</li>
		<li>Soak the dissection instruments in 70% ethanol for sterilization.</li>
		<li>Euthanize donor mice by carbon dioxide (CO2) inhalation followed by cervical dislocation, then spray the mice with 70% ethanol at the sites of manipulation.</li>
		<li>Use sterile dissection instruments to extract the femur, tibia and fibula bones without cutting any bone epiphyses.</li>
		<li>While securing the bones with a forceps, scrape off all tissue from bones (and discard fibula) using sterile scissors (or scalpel blades). Place bones in PBS on ice until all bones are collected.</li>
		<li>Carry out all remaining steps in sterile tissue culture hood. Using sterile instruments, secure bones with forceps and cut off both epiphyses to expose the medullary cavity.</li>
		<li>Using 3 ml syringes and 30 G needles, and cold flushing medium (Dulbecco Modified Eagle Medium [DMEM] supplemented with 10% fetal calf serum [FCS, heat-inactivated], 2 mM L-glutamine, 1% antibiotic-antimycotic solution, 50 &#181;M &#914;-mercaptoethanol), flush the red bone marrow into a Petri dish.</li>
		<li>Use the same syringe to dissociate flushed bone marrow cell clusters by repeated gentle aspiration and ejection.</li>
		<li>Pool femoral and tibial bone marrow from each mouse into one 15 ml centrifuge tube. Fill the tube with cold flushing medium to wash bone marrow cells and centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>BMCMCs Culture.
	<ol>
		<li>Remove supernatant and recover pelleted bone marrow cells in 10 ml of culture medium (DMEM supplemented with 10% fetal calf serum [FCS, heat-inactivated], 2 mM L-glutamine, 1% antibiotic-antimycotic solution, 50 &#181;M &#914;-mercaptoethanol and 20% WEHI-3 cell-conditioned medium [as a source of IL-3; alternatively use recombinant mouse IL-3 at 10 ng/ml]).</li>
		<li>Place cells in tissue culture flask of appropriate size (e.g., T25 for 1 mouse, T75 for 2 mice, etc.).</li>
		<li>1-2 days after plating cells, transfer medium and suspension cells (leaving debris and adherent cells sticking to bottom of flask) to a new flask and add fresh culture medium (10 ml/mouse).</li>
		<li>During the following weeks, feed cells every 3-4 days (add 10 ml of culture medium per mouse). Maintain cell density between 2.5 x 105-1 x 106 cells/ml. Transfer non-adherent cells to a new flask once a week until no adherent cells are present in the culture flask. Test maturity of cells (see below) before use for in vitro assays or engraftment into MC-deficient mice. 
		NOTE: Complete differentiation of BMCMCs will take 4-6 weeks.</li>
	</ol>
	</li>
	<li>Assessment of BMCMC maturity.
	<ol>
		<li>Using flow cytometry.
		<ol>
			<li>Wash cells (5 x 104-5 x 105 cells/condition) with ice-cold FACS buffer (PBS, 0.5% FCS) in 5 ml polystyrene round bottom tube. Centrifuge at 400 x g for 5 min at 4 &#176;C. Aspirate supernatant.</li>
			<li>Dilute anti-mouse CD16/32 (clone 93 or 2.4G2) monoclonal antibodies 1:200 (2.5 &#956;g/ml) in FACS buffer. Add 10 &#181;l to each pellet and resuspend by brief vortexing. Incubate 5 min on ice to block Fc binding. 
			NOTE: Protect samples from direct light for all remaining steps.</li>
			<li>Dilute phycoerythrin (PE) conjugated anti-mouse Fc &#949;RI &#945; (clone MAR-1) 1:200 (1 &#956;g/ml) and fluorescein isothiocyanate (FITC) conjugated anti-mouse KIT (CD117; clone 2B8) 1:200 (2.5 &#956;g/ml) (&#8220;staining solution&#8221;), or the respective labeled isotype control antibodies in FACS buffer (&#8220;isotype control solution&#8221;).</li>
			<li>Split half of the blocked cells from step 1.3.1.2) into an additional polystyrene round bottom tube and add 20 &#181;l of &#8220;staining solution&#8221; in one tube and 20 &#181;l of &#8220;isotype controls solution&#8221; in the other tube. Incubate 30 min on ice.</li>
			<li>Add 3 ml ice-cold FACS buffer and centrifuge at 400 x g for 5 min at 4 &#176;C. Discard supernatant and resuspend the pellet in 300 &#181;l FACS buffer containing 1 &#181;g/ml propidium iodide (PI, for identification of dead cells). Proceed to flow cytometry analysis (see Figure 2A).</li>
		</ol>
		</li>
		<li>Using toluidine blue staining.
		<ol>
			<li>Wash 1 x 105 cells once with PBS by centrifugation at 400 x g for 5 min at room temperature, then aspirate and resuspend cells in 200 &#181;l of PBS.</li>
			<li>Transfer the cell suspension into a prepared cytofunnel attached to a microscope slide and proceed with cyto-centrifugation using a cytocentrifuge (40 x g for 5 min).</li>
			<li>Air dry slides for 10 min and draw a wax circle around cells using a PAP pen. Add 0.1% Toluidine blue solution to cover the cells. Incubate for 1 min. Wash slides in running tap water for 1 min, then air-dry slides for 10 min.</li>
			<li>Coverslip cells using mounting medium. Proceed to light microscope analysis (see Figure 2B)</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Engraftment of Mast Cell-deficient Mice with BMCMCs.
<ol>
	<li>Ear engraftment by intradermal (i.d.) injection. 
	NOTE: For adoptive transfer of BMCMCs into the ear pinnae of MC-deficient mice, we recommend performing two injections of 1 x 106 cells each (for a total of 2 x 106 cells) per ear pinna. Intradermal engraftment of BMCMCs into the ear pinnae of MC-deficient mice has been used by many investigators in several models, including models of passive cutaneous anaphylaxis (PCA)24,27, host defense against venoms28, and chronic hypersensitivity (CHS) reactions29,30.
	<ol>
		<li>Count and resuspend cells at 4 x 107 BMCMCs/ml (1 x 106 BMCMCs/ 25 &#181;l) in cold DMEM. Transfer BMCMCs solution into a 1 ml syringe equipped with a 30 G needle. Keep on ice until injection.</li>
		<li>Anesthetize 4-6 weeks old MC-deficient mice using isoflurane (2.5% v/v). Check depth of anesthesia by toe pinch, adjust isoflurane if indicated and continue to monitor breathing and toe pinch response throughout the procedure. Apply ophthalmic ointment with a Q-tip to prevent dryness of eyes.</li>
		<li>With the index finger, create vertical pressure on the dorsal face of the ear pinna to expose and stretch the ventral face.</li>
		<li>Perform two 25 &#181;l i.d. injections of BMCMC solution into two different sites of the ventral face of the ear pinna, the first injection in the middle of the ear and the second injection toward the tip of the ear pinna. Wait 4-6 weeks after i.d. engraftment before performing in vivo experiments. 
		Note: In our experience, by that time the number of MCs/mm2 of dermis in the central part of the ear pinna generally is similar to that in the corresponding location in wild type mice, whereas the numbers of MCs/mm2 in the periphery of the ear pinnae are typically substantially lower than those in the corresponding wild type mice27,28. This should be kept in mind when designing experiments with such MC-engrafted mice (e.g., agents with possible effects on MC function should be injected into the central part of the ear pinnae, and the analysis of the effects of such treatments should also focus on that area).</li>
	</ol>
	</li>
	<li>Peritoneal cavity engraftment by intraperitoneal (i.p.) injection. 
	NOTE: Adoptive transfer of BMCMCs into the peritoneal cavity of MC-deficient mice will require one injection of 2 x 106 cells per mouse. Such i.p. injections of BMCMCs into MC-deficient mice have been used by many groups to study the roles of peritoneal MCs in various models, including models of host defense against venoms31 or bacterial sepsis32,33.
	<ol>
		<li>Count appropriate number of cells and resuspend at 1 x 107 BMCMCs/ml (2 x 106 BMCMCs/ 200 &#181;l) in cold DMEM. Transfer BMCMCs solution into a 1 ml syringe equipped with a 25 G needle. Keep on ice until injection.</li>
		<li>Perform 1 injection of 200 &#181;l BMCMCs solution into the peritoneal cavity of 4-6 week old MC-deficient mice. Wait 4-6 weeks after i.p. engraftment before performing in vivo experiments.</li>
	</ol>
	</li>
	<li>Engraftment by intravenous (i.v.) injection. 
	NOTE:Adoptive transfer of BMCMCs by i.v. injection into MC-deficient mice will require one injection of 5 x 106 cells per mouse. Intravenous (i.v.) injections of BMCMCs into MC-deficient mice have been used by many groups to study the roles of MCs in various disease models, including models of bladder infection34, asthma35, lung fibrosis36, and antibody-mediated arthritis37.
	<ol>
		<li>Count appropriate number of cells and resuspend at 2.5 x 107 BMCMCs/ml (5 x 106 BMCMCs/200 &#181;l) in cold DMEM. Transfer BMCMCs solution into a 1 ml syringe equipped with a 30 G needle. Keep on ice until injection.</li>
		<li>Anesthetize 4-6 weeks old MC-deficient mice using isoflurane (2.5% v/v). Check depth of anesthesia by toe pinch, adjust isoflurane if indicated and continue to monitor breathing and toe pinch response throughout the procedure. Apply ophthalmic ointment with a Q-tip to prevent dryness of eyes.</li>
		<li>Perform one injection of 200 &#181;l of BMCMC solution into the tail vein (or, alternatively, the retro-orbital vein) of a MC-deficient mouse. Wait 12 weeks after i.v. engraftment before performing in vivo experiments.</li>
	</ol>
	</li>
</ol>
3. Analysis of Engrafted Mast Cell-deficient Mice.
<ol>
	<li>Ear engraftment analysis.
	<ol>
		<li>At the end of the experiment, euthanize mice by CO2 inhalation followed by cervical dislocation.</li>
		<li>Isolate the ear pinnae and fix in 10% (vol/vol) buffered formalin overnight at 4 &#176;C. Embed fixed ear pinnae in paraffin, cut 4 &#181;m sections of ear pinnae and mount on glass slides.</li>
		<li>Stain the slides with a 0.1% Toluidine blue solution for 1 min at room temperature. Wash slides in tap water for 1 min, then air-dry slides for 10 min at room temperature.</li>
		<li>Coverslip slides using mounting medium, then count the number of engrafted MCs per pinnae sections using a light microscope. Evaluate the engraftment efficiency by comparing the percentage and distribution of MCs in the ear skin of engrafted mice versus wild type mice.</li>
	</ol>
	</li>
	<li>Peritoneal cavity engraftment analysis.
	<ol>
		<li>Evaluation of mast cells number in the peritoneal cavity.
		<ol>
			<li>At the end of the experiment, euthanize mice by CO2 inhalation followed by cervical dislocation. Remove carefully the ventral skin of the mice without breaking the peritoneal cavity.</li>
			<li>Inject 5 ml of cold or room temperature PBS in the peritoneal cavity using a 5 ml syringe equipped with a 25 G needle. Use cold PBS to reduce the risk of activating peritoneal cells (this is very important when evaluating peritoneal MC degranulation or levels of some MC-derived products in the peritoneal lavage fluid). Perform a massage of the abdomen for 20 sec to harvest peritoneal cells.</li>
			<li>Slowly aspirate the peritoneal lavage using a 5 ml syringe equipped with a 22 G needle. Record the volume of aspirated lavage (expect to recover up to 80% of injected volume).</li>
			<li>Transfer peritoneal lavage fluid into a 5 ml polystyrene round bottom tube and centrifuge at 400 x g for 5 min at 4 &#176;C. Aspirate supernatant. Recover the pellet in 400 &#181;l cold PBS.</li>
			<li>Count total number of peritoneal cells using a hemocytometer chamber. Use half of the cell suspension for a flow cytometry analysis (as described in step 1.3.1), to evaluate the percentage and marker expression of engrafted MCs in the peritoneal cavity. Multiply the total number of peritoneal cells by the percentage of MCs to obtain absolute number of peritoneal MCs.</li>
			<li>Perform a cytocentrifugation with the remaining cells as described in step 1.3.2.2. Air dry slides for 10 min at room temperature and draw a wax circle around cells using a PAP pen.</li>
			<li>Cover cells with undiluted May-Gr&#252;nwald Staining solution for 5 min, followed by washing in PBS for 5 min, both at room temperature. Cover cells with Giemsa stain diluted 1:20 with deionized water and incubate 20 min at room temperature. Wash slides 2 times in tap water for 1 min, then air-dry slides.</li>
			<li>Coverslip cells using mounting medium, and calculate the percentage of engrafted MCs using a light microscope (by counting at least 400 total cells). Evaluate the engraftment efficiency by comparing the percentage of MCs in the peritoneal lavage of engrafted mice versus wild type mice.</li>
		</ol>
		</li>
		<li>Evaluation of mast cells in the mesenteric windows.
		<ol>
			<li>Following the peritoneal lavage described in step 3.2.1, cut open the peritoneal membrane to expose the intestinal tract of the mice. Arrange 4 to 5 mesenteric windows per mouse onto a slide.</li>
			<li>Fix the slides for 1 hr in Carnoy solution (3:2:1 vol/vol/vol of ethanol, chloroform, and glacial acetic acid) at room temperature. Air-dry slides and remove the intestine (the fixed mesenteric windows will remain attached to the slides).</li>
			<li>Stain the preparations for 20 min at room temperature with Csaba (Alcian blue/Safranin O) staining solution.
			<ol>
				<li>To make 500 ml of Csaba stain, prepare 500 ml of acetate buffer by mixing 100 ml of 1 M sodium acetate solution with 120 ml of 1 M HCl. Make up to 500 ml with deionized water and adjust pH to 1.42. Then, dissolve 90 mg Safranin [identifies &#8216;mature MCs&#8217; in red], 1.8 g Alcian blue [identifies &#8216;immature MCs&#8217; in blue] and 2.4 g ferric ammonium sulphate NH4Fe(SO4)2 into 500 ml of acetate buffer.</li>
			</ol>
			</li>
			<li>Coverslip slides using mounting medium, then count the number of engrafted MCs per mesenteric window using a light microscope. Evaluate the engraftment efficiency by comparing the percentage and distribution of MCs in the mesenteric windows of engrafted mice versus wild type mice.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>I.v. engraftment analysis.
	<ol>
		<li>At the end of the experiment, euthanize mice by CO2 inhalation followed by cervical dislocation, and harvest tissue of interest (e.g. lung, skin or spleen) and fix overnight in 10% (vol/vol) buffered formalin at 4 &#176;C.</li>
		<li>Embed fixed tissues in paraffin, cut 4 &#181;m sections, and mount on glass slides. Stain the slides with a 0.1% Toluidine blue solution for 1 min at room temperature. Wash slides in tap water for 1 min, then air-dry slides for 10 min.</li>
		<li>Coverslip slides using mounting medium and evaluate number and distribution of engrafted MCs per tissue sections using a light microscope.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Almost 30 years after its initial description38, the &#8216;mast cell knock-in&#8217; approach continues to provide valuable information about what MCs can do or can&#8217;t do in vivo. The functions of MCs were long thought to be limited to their role in allergy. Data generated using the &#8216;mast cell knock-in&#8217; approach have changed this view, by providing evidence that MCs can, among other functions, play critical roles in host defense against certain pathogens4,39 or venoms28,31, or can even suppress certain immune responses29,34,40.
In our protocol description, we decided to focus on the generation and engraftment of bone marrow-derived cultured MCs (BMCMCs), because large numbers of these cells can be generated in vitro from the bone marrow of wild type or mutant mice. However, MCs can also be cultured directly from embryonic stem cells (embryonic stem cell-derived cultured MCs [ESCMCs])41 and, when genetically compatible, these cells can also be used for engraftment into MC-deficient mice. This alternative approach is particularly interesting for studying the role of a protein whose deficiency induces embryonic lethality in mice, and therefore for which BMCMCs deficient for this protein cannot be generated. Both BMCMCs and ESCMCs can also be transduced in vitro with lentiviruses encoding genes of interest or shRNA to silence genes of interest, before engraftment of these cells into MC-deficient mice31,33.
We usually use 20% WEHI-3-conditioned medium as a source of IL-3 for the culture of BMCMCs. However, recombinant IL-3 (10 ng/ml, as described in step 1.2.1) can also be used, and addition of recombinant stem cell factor (SCF) to the culture medium can substantially enhance the numbers of BMCMCs generated42,43. Depending on the study, 10 to 100 ng/ml of recombinant SCF have been used, in addition to IL-3, to generate BMCMCs36,44,45. One should keep in mind that commercially available murine recombinant SCF preparations from different suppliers may differ in their potency in influencing the development of BMCMCs. It is also important to recognize that details of approach used to generate BMCMCs (such as whether one adds recombinant SCF to IL-3-containing medium, the duration of the culture period, etc.) may influence the phenotype and function of such cells. For example, it has been reported that BMCMCs chronically exposed to SCF have increased levels of histamine and certain proteases44,46, but display a marked attenuation of Fc&#949;RI-mediated degranulation and cytokine production in vitro45. Finally, in addition to IL-3, WEHI-3-conditioned medium contains many biologically active molecules that may affect MC functions. BMCMCs obtained with WEHI-3-conditioned medium are therefore likely to differ from BMCMCs obtained with recombinant IL-3 (or with recombinant IL-3 plus SCF). There have been few studies of whether or for how long any such differences in the phenotypes of BMCMCs generated in different types of culture medium are retained after the cells&#8217; engraftment into different anatomical sites in vivo, and additional studies of this type may be of interest. However, regardless of the chosen culture conditions, the same culture medium recipe should be used to generate all of the BMCMCs to be used for engraftment in experiments from which results will be pooled for analysis. Moreover, MCs should be cultured for at least 4 to 6 weeks before their engraftment into MC-deficient mice, in order to reach a purity of 95-98% (Figure 2). This is to reduce the possibility that the presence, in the &#8220;BMCMC populations&#8221;, of hematopoietic cells other than those committed to the mast cell lineage (which are present in the cultures at early intervals after placing the bone marrow cells in vitro) might result in the appearance of donor-derived cells in addition to MCs in the &#8216;mast cell knock-in&#8217; mice.
We present here a detailed protocol for engrafting MC-deficient mice with wild type or mutant BMCMCs intraperitoneally (i.p.), intravenously (i.v.) or intradermally (i.d.) in the ear pinna, since these routes of injection have been used by many investigators. However, BMCMCs have also been successfully engrafted into the back skin47, in the footpad48, intra-articularly49 or intra-cranially50,51. The number of BMCMCs to engraft, as well as the interval between engraftment and experiment, can vary depending on the route of injection and the targeted organ to engraft (Figure 1). It is very important to respect such intervals after engrafting the BMCMCs before starting the experiment in order to allow sufficient time for BMCMCs (which are not fully mature MCs) to become more mature in vivo. Because the content of mediators stored in the MC&#8217;s cytoplasmic granules can continue to increase during the course of the cell&#8217;s lifetime38,52, for certain experiments one may wish to increase the interval between MC engraftment and the initiation of the experiment to assess MC function.
Depending on the route of injection and/or the numbers of BMCMCs injected, the numbers and/or anatomical distribution of the adoptively transferred MCs can differ from those of the corresponding native MC populations in wild type mice12,23,53,54. MC-deficient mice engrafted i.p. or i.d. with BMCMCs can have about the same numbers and distribution of MCs than the native MC population in wild type mice, in the peritoneal cavity and mesentery and in the dermis, respectively when assessed 4 to 8 weeks after MC transfer12,23. Intravenous transfer of BMCMCs does not lead to normal MC numbers and/or distribution in most tissues. For example, no or very few MCs are found in the skin of such i.v.-engrafted &#8216;mast cell knock-in&#8217; mice. At 4-28 weeks after i.v. injection of BMCMCs into MC-deficient mice, numbers of MCs in the trachea are substantially lower than those in the corresponding wild type mice. By contrast, the numbers of MCs in the periphery of the lung are typically greater than those in the corresponding wild type mice12,23,53,55. I.v. transfer of BMCMCs also results in high levels of MCs in the spleen, whereas very few native MCs are typically found in this organ in wild type mice23,56. Importantly, previous reports demonstrated that i.v. injection of BMCMCs into MC-deficient mice fails to result in engraftment of the MC populations in specific anatomical sites such as the spinal cord, lymph nodes or heart54,57. Several groups have also noted that such i.v. engraftment does not result in engraftment of the intestinal mucosal MC (MMC) population23,58-60. Such differences in MC numbers and/or distribution of adoptively-transferred MCs versus native MCs must be taken into account when interpreting data obtained using the MC &#8216;mast cell knock-in&#8217; model9.
Several strains of MC-deficient mice exist, and choosing which one(s) to use in a particular project is important. c-kit mutant MC-deficient mice, such as KitW/W-v or KitW-sh/W-sh mice, have been traditionally used by many investigators. However, such mice suffer from many c-kit-related phenotypic abnormalities beside their profound MC deficiency (Table 1). In recent years, several strains of mice with c-kit-independent constitutive MC deficiency have been reported24-26. Some of these mice also exhibit other phenotypic abnormalities beside their MC deficiency (Table 1), and additional abnormalities might also be discovered as the phenotype of these newly described strains is still under investigation. All these mice and some additional new types of MC-deficient mice have been recently reviewed in detail9,10,13.
Given the limitations of each of the MC-deficient strains currently available, we recommend attempting to test hypotheses about MC function using more than one model of MC deficiency9. In our laboratory, we generally perform pilot experiments in KitW-sh/W-sh and Cpa3-Cre; Mcl-1fl/fl mice. If we obtain concordant results in both types of MC-deficient mice, we then proceed to engraftment experiments to ascertain the role of MCs and assess the potential roles of certain MC-derived products. Finally, it should be noted that several strains allowing inducible depletion of MCs or Cre recombinase-mediated deletion of &#8220;floxed&#8221; genes in MCs have also been recently described25,49,61. These strains have been reviewed in detail elsewhere9,10,13 and represent promising alternative - or complementary - approaches to study MC functions in vivo.

Mast cells (MCs) are hematopoietic cells that arise from pluripotent bone marrow progenitors1-3. Following bone marrow egression, MCs progenitors migrate into various tissues where they develop into mature MCs under the influence of local growth factors1-3. Tissue-resident MCs are strategically located at host-environment interfaces, such as the skin, the airways and the gastrointestinal tract, where they behave as a first line of defense against external insults3-6. MCs are often sub-classified based on their &#8220;baseline&#8221; phenotypic characteristics and their anatomic locations. In mice, two types of MCs have been described: &#8220;connective tissue-type&#8221; MCs (CTMCs) and mucosal MCs (MMCs)1-3,7,8. CTMCs are often located around venules and near nerve fibers, and reside in serosal cavities, while MMCs occupy intraepithelial locations in the gut and respiratory mucosa1-3.
Numerous methodologies have been applied to study biological functions of MCs9-13. Many groups have focused on in vitro approaches using either cell lines (such as the human MC lines HMC114 or LAD215,16), in vitro derived MCs (such as human peripheral blood-derived MCs17, or mouse bone marrow-derived cultured MCs [BMCMCs]18, fetal skin-derived cultured MCs [FSCMCs]19 and peritoneal cell-derived MCs [PCMCs]20) or ex vivo isolated MCs from different anatomical sites. All these models are widely used to study molecular details of MC biology, such as signaling pathways involved in MC activation. However, an important aspect of MCs biology is that their phenotypic and functional characteristics (e.g., cytoplasmic granule protease content or response to different stimuli) can be modulated by anatomical location and microenvironment2,7. Since the exact mixture of such factors that are encountered in vivo may be difficult to reproduce in vitro, we favor using in vivo approaches to gain insights into MCs functions9.
Several mouse strains with genetic MC deficiency exist, such as the widely used WBB6F1-Kit W/W-v or C57BL/6-Kit W-sh/W-sh mice. These mice lack expression and/or activity of KIT (CD117), the receptor for the main MC growth factor stem cell factor (SCF)21,22. As a result, these mice have a profound MC deficiency but also have additional phenotypic abnormalities related to their c-kit mutations (in the WBB6F1-Kit W/W-v mice) or to the effects of the large chromosomal inversion that results in reduced c-kit expression (in the C57BL/6-Kit W-sh/W-sh mice)9,10,12,23. More recently, several strains of mice with c-kit-independent constitutive MC deficiency have been reported24-26. All these mice and some additional new types of inducible MC-deficient mice have been recently reviewed in detail9,10,13.
Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This so-called &#8216;mast cell knock-in&#8217; method can be used to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

<table><tbody><tr><td>1% Antibiotic-Antimycotic Solution</td><td>Corning cellgro</td><td>30-004-Cl</td><td></td></tr><tr><td>3 ml Syringe</td><td>Falcon</td><td>309656</td><td></td></tr><tr><td>35 mm x 10 mm Dish</td><td>Corning cellgro</td><td>430588</td><td></td></tr><tr><td>5 ml Polystyrene Round Bottom Tube</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>Acetic Acid Glacial</td><td>Fisher Scientific</td><td>A35-500</td><td></td></tr><tr><td>Alcian Blue 8GX</td><td>Rowley Biochemical Danver</td><td>33864-99-2</td><td></td></tr><tr><td>Allegra 6R Centrifuge</td><td>Beckman</td><td></td><td></td></tr><tr><td>Anti-mouse CD16/32 (clone 93) Purified</td><td>eBioscience</td><td>14-0161-81</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma Aldrich</td><td>M7522</td><td></td></tr><tr><td>BD 1 ml TB Syringe</td><td>BD Syringe</td><td>309659</td><td></td></tr><tr><td>BD 22 G x 1 (0.7 mm x 25 mm) Needles</td><td>BD Precision Glide Needle</td><td>205155</td><td></td></tr><tr><td>BD 25 G 5/8 Needles</td><td>BD Syringe</td><td>305122</td><td></td></tr><tr><td>BD 30 G x 1/2 Needles</td><td>BD Precision Glide</td><td>305106</td><td></td></tr><tr><td>Blue MAX Jr, 15 ml Polypropylene Conical Tube</td><td>Falcon</td><td>352097</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C298-500</td><td></td></tr><tr><td>Cytoseal 60 Mounting Medium</td><td>Richard-Allan Scientific</td><td>8310-4</td><td></td></tr><tr><td>Cytospin3</td><td>Shandon</td><td>NA</td><td></td></tr><tr><td>DakoCytomation pen</td><td>Dako</td><td>S2002</td><td></td></tr><tr><td>Dulbecco Modified Eagle Medium (DMEM) 1x</td><td>Corning cellgro</td><td>15-013-CM</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>E 7023-500ml</td><td></td></tr><tr><td>Fetal Bovine Serum Heat Inactivated</td><td>Sigma Aldrich</td><td>F4135-500ml</td><td></td></tr><tr><td>FITC Conjugated IgG2b K Rat Isotype Control</td><td>eBioscience</td><td>14-4031-82</td><td></td></tr><tr><td>Fluorescein Isotiocyanate (FITC) Conjugated Anti-mouse KIT (CD117; clone 2B8)</td><td>eBioscience</td><td>11-1171-82</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher Scientific</td><td>F79-500</td><td></td></tr><tr><td>Giemsa Stain Modified</td><td>Sigma Aldrich</td><td>GS-1L</td><td></td></tr><tr><td>Isothesia</td><td>Henry Schein Animal Health</td><td>29405</td><td></td></tr><tr><td>May-Grunwald Stain</td><td>Sigma Aldrich</td><td>MG-1L</td><td></td></tr><tr><td>Multiwell 6 well plates</td><td>Falcon</td><td>35 3046</td><td></td></tr><tr><td>Olympus BX60 Microscope</td><td>Olympus</td><td>NA</td><td></td></tr><tr><td>Paraplast Plus Tissue Embedding Medium</td><td>Fisher Brand</td><td>23-021-400</td><td></td></tr><tr><td>PE Conjugated IgG Armenian Hamster Isotype Control</td><td>eBioscience</td><td>12-4888-81</td><td></td></tr><tr><td>Phosphate-Buffered-Saline (PBS) 1x</td><td>Corning cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>Phycoerythrin (PE) Conjugated Anti-mouse FceRIa (clone MAR-1)</td><td>eBioscience</td><td>12-5898-82</td><td></td></tr><tr><td>Propidium Iodide Staining Solution</td><td>eBioscience</td><td>00-6990-50</td><td></td></tr><tr><td>Recombinant Mouse IL-3</td><td>Peprotech</td><td>213-13</td><td></td></tr><tr><td>Safranin-o Certified</td><td>Sigma Aldrich</td><td>S8884</td><td></td></tr><tr><td>Tissue culture flasks T25 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Beckton Dickinson</td><td>353109</td><td></td></tr><tr><td>Tissue culture flasks T75 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Beckton Dickinson</td><td>353110</td><td></td></tr><tr><td>Toluidine Blue 1% Aqueous</td><td>LabChem-Inc</td><td>LC26165-2</td><td></td></tr><tr><td>Recombinant Mouse SCF</td><td>Peprotech</td><td>250-03</td><td></td></tr></tbody></table>

analyzing the functions of mast cells in vivo using 'mast cell knock-in' mice

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the &#8216;mast cell knock-in&#8217; approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

An overview of the &#8216;mast cell knock-in&#8217; approach is shown in Figure 1, and includes the generation of BMCMCs, the number of cells that should be engrafted i.p., i.d. or i.v. into MC-deficient mice (the number can be varied if indicated based on the experimental design) and the interval between engraftment and experiment depending on the injection site (this interval also can vary, if indicated; e.g., the content of stored mediators in MC cytoplasmic granules increases steadily with time38). Figure 2 shows representative flow cytometry analyses and toluidine blue staining of BMCMCs after 1, 15 and 45 days of culture in DMEM medium containing in 20% WEHI-3 cell-conditioned medium as a source of IL-3. Note that BMCMCs cultured for 45 days are 95% pure, contain high numbers of cytoplasmic granules and can be used for engraftment experiments, while cells cultured for 15 days are not suitable for engraftment. Figure 3 shows representative successful engraftment in the ear pinnae 4 weeks after i.d. engraftment (Figure 3A), in mesenteric windows (Figure 3B) and peritoneal cavity (Figure 3C) 6 weeks after i.p. engraftment, and in the lung (Figure 3D) 12 weeks after i.v. engraftment.
<img alt="Figure 1" src="/files/ftp_upload/52753/52753fig1.jpg" /> 
Figure 1: &#8216;Mast cell knock-in&#8217; mouse model for analyses of MC functions in vivo. Wild type or mutant bone marrow-derived cultured MCs (BMCMCs) are generated by culturing bone marrow cells for at least 4-6 weeks in 20% WEHI-3 cell-conditioned medium as a source of IL-3 (or alternatively in medium containing 10 ng/ml recombinant mouse IL-3). These BMCMCs can then be engrafted into MC-deficient mice to create so-called &#8216;mast cell knock-in&#8217; mice. BMCMCs can be injected via different routes (intravenous [i.v.], intraperitoneal [i.p.] or intradermal [i.d.]) for local (i.d., i.p.) or systemic (i.v.) reconstitution of various MC populations. MC function(s) in various biological responses can then be analyzed by comparing the responses in wild type mice, MC-deficient mice and &#8216;mast cell knock-in&#8217; mice. The contribution(s) of specific MC products can be analyzed by comparing responses of &#8216;mast cell knock-in&#8217; mice engrafted with either wild type BMCMCs or BMCMCs derived from mice that lack, or express genetically-altered forms of, such products. (This is a modified version of Figure 1 from ref.12).
<img alt="Figure 2" src="/files/ftp_upload/52753/52753fig2.jpg" /> 
Figure 2: Evaluation of the purity of BMCMC preparations by flow cytometry and microscopy. (A) Representative flow cytometry analyses of Fc&#949;RI&#945; and KIT (CD117) expression on the surface of 1 day (left panel), 15 days (middle panel) and 45 days (right panel) old BMCMCs. Propidium iodide (PI)-positive dead cells were excluded from the analysis (not shown). Numbers indicate the percentage of Fc&#949;RI&#945;+KIT+ BMCMCs gated in the blue square. (B) Representative pictures of BMCMCs stained with toluidine blue, lower panel is a magnification of the regions of the upper panel defined in black dotted lines. Bars = 10 &#956;m.
<img alt="Figure 3" src="/files/ftp_upload/52753/52753fig3.jpg" /> 
Figure 3: Evaluation of the efficiency of MC engraftment at various anatomical sites. (A) Representative pictures of 4 &#956;m ear pinnae sections stained with toluidine blue. (B) Representative pictures of mesenteric windows stained with Safranin/Acian Blue (&#8216;Csaba&#8217; stain). (C) Representative pictures of cells present in peritoneal lavage fluids and stained with May-Gr&#252;nwald Giemsa (MGG). (D) Representative pictures of 4 &#956;m lung sections stained with toluidine blue. Bars = 50 &#956;m (A, B and D) or 20 &#956;m (C). Pictures are from C57BL/6 wild type mice (upper panel), MC-deficient KitW-sh/W-sh mice (middle panel) and MC-deficient mice engrafted with wild type BMCMCs: BMCMCs KitW-sh/W-sh (lower panel). MCs are indicated with arrows.
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mice</td>
			<td>Available Backgrounds</td>
			<td>MC numbers 
			(steady state)</td>
			<td>Other phenotypes (reviewed in9,10,13)</td>
			<td>Reported engraftments with BMCMCs</td>
		</tr>
		<tr>
			<td>KitW/W-v</td>
			<td>(WB/Re x C57BL/6)F1 
			(Jackson Laboratories &#8211; 
			WBB6F1/J-KitW/KitW-v/J)</td>
			<td>Absence of connective-tissue and mucosal MCs</td>
			<td>Anemia, reduced basophil and neutrophil numbers, deficiencies in melanocytes and interstitial cells of Cajal, sterile, etc.</td>
			<td>i.v.35,37; i.p.31,62; i.d.28,29; i.c.50,51; i.a.49; fp.i.63</td>
		</tr>
		<tr>
			<td rowspan="3">KitW-sh/W-sh</td>
			<td>C57BL/6 
			(Jackson Laboratories &#8211; 
			B6.Cg-KitW-sh/HNihrJaeBsmJ) Single nucleotide polymorphism analysis performed at Jackson laboratories shows that these mice are only ~87% C57BL/6-genetic background.</td>
			<td rowspan="3">Absence of connective-tissue and mucosal MCs</td>
			<td rowspan="3">Moderate increase in basophil and neutrophil numbers, increased numbers of myeloid-derived suppressor cells64, deficiencies in melanocytes and interstitial cells of Cajal</td>
			<td rowspan="3">i.v.23,34-36; i.p.31,62; i.d.28,29; i.a.49</td>
		</tr>
		<tr>
			<td>C57BL/6J62 
			(Jackson Laboratories &#8211; 
			B6.Cg-KitWsh/HNihrJaeBsmGlliJ) These mice have been backcrossed &#62;11 times on the C57BL/6J background.</td>
		</tr>
		<tr>
			<td>BALB/c65 
			(C.B6-KitW-sh)</td>
		</tr>
		<tr>
			<td>Mcpt5-Cre; 
			R-DTA</td>
			<td>C57BL/625 
			(Tg(Cma1-cre) ARoer; B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky/J)</td>
			<td>Marked reductions in peritoneal (98%) and skin (89-96.5%) MCs, mucosal MCs unlikely to be depleted</td>
			<td>Probable presence of mucosal MCs; reporter mice reveals Cre-mediated deletion of &#8216;floxed&#8217; YFP transgene in ~30% spleen NK cells66</td>
			<td>none</td>
		</tr>
		<tr>
			<td rowspan="2">Cpa3Cre/+</td>
			<td>C57BL/626 
			(B6.129P2-Cpa3tm3(icre)Hrr)</td>
			<td rowspan="2">Absence of connective-tissue and mucosal MCs</td>
			<td rowspan="2">Cpa3 expressed in other cell types; reduced basophil numbers</td>
			<td rowspan="2">i.v.26</td>
		</tr>
		<tr>
			<td>BALB/c26 
			(B6.129P2/OlaHsd.BALB/c-Cpa3tm3(icre)Hrr)</td>
		</tr>
		<tr>
			<td>Cpa3-Cre; 
			Mcl-1fl/fl</td>
			<td>C57BL/624 
			(Tg(Cpa3-cre)3Glli; B6;129-Mcl1tm3sjkJ)</td>
			<td>Absence of connective-tissue and mucosal MCs</td>
			<td>Cpa3 expressed in other cell types; increased spleen neutrophils &#38; mild anemia; 
			reduced basophil numbers</td>
			<td>i.d.24; 
			i.a.49 
			i.v. (our unpublished data)</td>
		</tr>
	</tbody>
</table>
Table 1: Strains of mice with constitutive MC deficiencies. Several strains of mice with c-kit-dependent or c-kit-independent constitutive MC deficiency are available. In principle, all of these strains can be used to generate &#8216;mast cell knock-in&#8217; mice (although, to the best of our knowledge, this has not yet been reported for Mcpt5-Cre;R-DTA mice). However, each of these strains has other phenotypic abnormalities and limitations that should be kept in mind when interpreting results obtained with these mice. Some examples of successful MC engraftment can be found in the references. (This is a modified and updated version of Table 1 from ref.9).

Watch this Scientific Journal Video about Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice at JoVE.com

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

analyzing-functions-mast-cells-vivo-using-mast-cell-knock

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.2K Views.  Stanford University School of Medicine. We beschrijven een methode voor het genereren van in vitro afgeleide mestcellen, hun engraftment in mastceldeficiënte muizen, en de analyse van het fenotype, aantallen en distributie van geïgrafeerde mestcellen op verschillende anatomische locaties. Dit protocol kan worden gebruikt om de functies van mestcellen in vivote beoordelen .

Video: Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, as visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse1.
Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-&kappa;B luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-&kappa;B signaling within the LPS-injected gland4.
These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse.

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as ...

Medicine

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Developmental Biology

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.

We have established an ex vivo mast cell degranulation assay carried out by incubating crude peritoneal exudate cells isolated from the mice, treated with a pharmacological agent of interest and administered anti-dinitrophenol (DNP) IgE beforehand, with DNP on a carrier protein.

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the ...

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

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