This work was supported by Faculty Research grants from the College of Arts and Sciences Dean's Office at the University of Hartford.

During the study, animal care and treatment complied with National Institutes of Health policy, were in accordance with institutional guidelines, and were approved by the University of Hartford Animal Institutional Animal Care and Use Committee.
1. Entrainment of Animals
NOTE: Twenty week-old male B6129SF2/J mice are used in the study.
<ol>
	<li>Entrain mice to a 12 h light (standard overhead white light)/12 h dark cycle for 2 weeks prior to the experiment. 
	NOTE: Here zeitgeber time (ZT) 0 corresponds to lights on and ZT12 to lights off, while keeping all other environmental factors (i.e., food, water, and room temperature) constant 21.</li>
</ol>
2. Preparation of Instruments, Culture Medium, and Challenge Medium
<ol>
	<li>Autoclave forceps and dissecting scissors. Wrap pairs of frosted microscope slides in aluminum foil and autoclave.</li>
	<li>Prepare culture medium by adding fetal bovine serum (FBS) to RPMI 1640 to a final concentration of 10%. Prepare 10 mL of challenge medium in 50 mL tubes by adding the following PAMPs to the culture medium (RPMI with 10% FBS); LPS (5 &#181;g/mL), ODN1826 (5 &#181;g/mL), heat-killed Listeria monocytogenes (108 HKLM/mL), or another PAMP in its suggested concentration.</li>
	<li>Warm culture medium and challenge medium to 37 &#176;C in a water bath, and keep approximately 70 mL of sterile phosphate buffered saline (PBS, pH 7.2) at room temperature.</li>
	<li>Add 10 mL of culture medium using a 10 mL pipette and pipette aid to a 50 mL tube and place on ice.</li>
	<li>Add approximately 30 mL of 70% ethanol to a 100 mL beaker and place ends of dissecting scissors and forceps into the beaker to prevent microbial contamination.</li>
	<li>Prepare lysis buffer for RNA isolation by adding 10 &#181;L &#946;-Mercaptoethanol (under a fume hood) to every 1 mL of Buffer RLT in a 50 mL tube. Make only the amount that will be needed (600 &#181;L per sample, which consists of approximately 1 x 106 cells). 
	NOTE: The Buffer RLT is a component of the RNA extraction kit that supports the binding of RNA to the silica membrane.</li>
</ol>
3. Splenocyte Isolation and Challenge
<ol>
	<li>Euthanize mice at a particular zeitgeber time via narcosis by keeping them in their original cage and adding CO2 to the cage at a flow rate of 3 L/min. Continue supplying CO2 for 1 min after breathing stops.</li>
	<li>Confirm death via cervical dislocation by placing the thumb and index finger on either side of the neck at the base of the skull. Alternatively, press a rod at the base of the skull while quickly pulling (using the other hand) the base of the tail or the hind limbs to cause separation of the cervical vertebrae from the skull22.</li>
	<li>Spray the mouse trunk with 70% ethanol and wipe with a paper towel. Place the mouse on its back and slightly tilted onto its right side. Cut away the fur, using dissecting scissors, along the mouse&#39;s left side, about halfway between the front and back legs.</li>
	<li>Using forceps, grab the peritoneum and carefully make an incision so as not to damage the spleen. Remove spleen with forceps and place into a sterile 50 mL tube containing approximately 10 mL of culture media on ice. Repeat for the remaining animals. 
	&#8203;NOTE: The spleen is the color of a kidney bean, and it is longer and flatter than the kidney.</li>
	<li>Transfer one spleen with 2 mL of culture medium to a small sterile Petri dish.
	<ol>
		<li>Homogenize the spleen by grinding it between the frosted portion of two sterile frosted slides. If possible, keep the issue and cells in the medium during the homogenization process.</li>
		<li>Once thoroughly homogenized, pipet the 2 mL of culture medium containing the splenocytes through a 40 &#181;m nylon cell strainer into a 50 mL tube.</li>
		<li>Repeat the above steps for the remaining spleens using new pairs of frosted slides, 50 mL tubes with culture medium, and cell strainers.</li>
	</ol>
	</li>
	<li>Add 8 mL of cold culture medium to each of the 50 mL tubes containing the splenocytes for a total volume of 10 mL/tube. Determine the number of cells per milliliter using a hemocytometer.</li>
	<li>Add approximately 1 x 106 cells/well to 6-well culture plates. Add cells to the number of wells that correspond to the number of different PAMPs being used for the experiment and include a control well. Add 3 mL of culture medium or 3 mL of challenge medium to the respective wells.</li>
	<li>Incubate the plates at 37 &#176;C in 5% CO2 for 3 h.</li>
	<li>Scrape the cells from the bottom of the well using a 1,000 &#181;L pipet tip attached to a P1000 micropipette. Transfer the medium containing the cells using the same 1,000 &#181;L pipet tip to a 15 mL tube.</li>
	<li>Pellet the cells via centrifugation at 167 x g for 5 min at room temperature. Remove the supernatant and wash the cell pellet with 5 mL of PBS.</li>
	<li>Pellet the cells a second time at 167 x g for 5 min, remove the supernatant, and add 600 &#181;L of lysis buffer to the cell pellet in order to lyse the cells. Then proceed to RNA isolation.</li>
</ol>
4. RNA Isolation and cDNA Synthesis
<ol>
	<li>Isolate RNA from splenocytes using the RNA extraction kit according to manufacturer&#39;s instructions and perform the &#39;optional&#39; on-column DNA digestion.</li>
	<li>Prior to cDNA synthesis, determine RNA concentration using a microvolume spectrophotometer to verify that the concentration is within the optimal RNA range (up to 2 &#181;g) of the cDNA synthesis kit.</li>
	<li>Synthesize cDNA for each of the samples using the reverse transcription kit according to manufacturer&#39;s instructions. Use 10 &#181;L of RNA for each of the samples in a 20 &#181;L total reaction volume. At the completion of the reverse transcription (thermocycler run) add 20 &#956;L H2O to each reaction.</li>
	<li>Construct the standard curving using a P10 or P20 micropipette to pool mRNA from a few control samples (e.g., 5 &#181;L from 2 samples for a total of 10 &#181;L) into a 0.5 mL tube to prepare the cDNA.
	<ol>
		<li>Add 10 &#181;L 2x reverse transcriptase master mix.</li>
		<li>At the completion of the reverse transcription, add 10 &#181;L of H2O to the reaction tube, which will serve as the starting concentration (&#34;1&#34;) in the dilution series for the standard curve.</li>
		<li>Perform a 10-fold dilution series (1 to 10-4) by adding 45 &#181;L of water using a P100 or P200 micropipette into four 0.5 mL tubes designated 10-1, 10-2, 10-3, and 10-4.
		<ol>
			<li>Add 5 &#181;L of the starting concentration &#34;1&#34; into the first tube (10-1) using a P20 micropipettor, mix by pipetting up and down several times, then transfer 5 &#181;L from the 10-1 tube into the tube designated 10-2 and mix.</li>
			<li>Transfer 5 &#181;L using a P20 micropipette from the 10-2 tube into the tube designated 10-3 and mix. Transfer 5 &#181;L using a P20 micropipettor from the 10-3 tube into the tube designated 10-4 and mix.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Quantitative Polymerase Chain Reaction (qPCR)
<ol>
	<li>Determine relative quantitation of mRNA levels by qPCR. Within the experimental set up, select &#34;Quantitation - Relative Standard Curve&#34; and &#34;TaqMan&#34; chemistry. Change the reaction volume to 10 &#181;L. Enter the relevant information into the plate layout (e.g., target gene, standard curve, reporter, etc.).</li>
	<li>Prepare the reaction so that it contains 0.5 &#181;L of the primer/probe assay, 5 &#181;L of gene expression master mix, 2 &#181;L of H2O and 2.5 &#181;L of cDNA (10&#8211;100 ng).
	<ol>
		<li>Prepare a master mix by multiplying each of the preceding reagents (except the cDNA) by the number of reactions, making sure to include those from the standard curve, negative controls, performing each reaction in duplicate, and 2 extra reactions that will account for pipetting variation.</li>
		<li>Pipet 7.5 &#181;L of the prepared master mix into pre-determined wells in a 96-well reaction plate with a micropipette or a multichannel pipette. Pipet 2.5 &#181;L of cDNA into the appropriate well for the unknowns and standards, and 2.5 &#181;L of H2O into the negative control wells.</li>
		<li>Include primer/probe assays for various molecular clock genes, and also include an assay for an endogenous control (e.g., &#946;-actin).</li>
	</ol>
	</li>
	<li>In order to determine relative expression values, calculate the mean relative quantity for each replicate, then, for each sample, divide the mean relative quantity for the target gene by the mean relative quantity for &#946;-actin.</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Using statistical analysis software, enter data into the program under the column statistics option. Select a one-way ANOVA with the Dunnett&#39;s post hoc test to assess differences between mean values of clock gene expression after PAMP challenge versus the control.</li>
</ol>

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The author has nothing to disclose.

Within this protocol, a microvolume spectrophotometer can be used to quantify and assess the purity of the RNA being used in determining gene expression. Nucleic acids absorb UV light at 260 nm, proteins typically absorb light at 280 nm, while other potential contaminants used during an RNA extraction procedure (e.g., phenol) are detectable at 230 nm. Therefore, by assessing the absorbance (A) ratio at 260/280 nm (RNA to protein) and 260/230 nm (RNA to non-protein contaminants) the quality of the RNA can be assessed. High quality RNA has an A260/280 ratio between 1.8&#8211;2.1, as lower ratios indicate protein contamination. A pure RNA sample will have an A260/230 ratio of 2.0.
When determining the relative expression of a target gene (i.e., Per2, Clock, Rev-erb&#945;, and Dbp), an endogenous control gene (a gene in which expression levels do not differ between samples) must also be selected. Relative expression of the target gene is then normalized to the expression of the endogenous control gene. Differences in starting material (number of splenocytes), variation in reverse-transcriptase efficiency, varying rates of RNA degradation, etc., will be corrected for by the endogenous control gene (&#946;-actin in this protocol). However, it is wise to verify that the treatment being examined does not alter expression of the endogenous control gene. This can be accomplished by assessing &#946;-actin levels from several replicates of an equal number of cells (treatment vs. non-treatment). In theory, their &#946;-actin levels should be identical. Another approach to guard against endogenous control variation would be to use a panel of endogenous controls (e.g., &#946;-actin, Gapdh, and 18S rRNA gene).
When examining the impact of PAMPs on the molecular clock, the time of day when mice are euthanized and splenocytes are subsequently challenged must be taken into consideration. Tlr expression and responsiveness has previously been shown to demonstrate time-of-day dependent variation8,15, therefore, a time of day when TLR responsiveness is at its peak, could result in a greater influence on the clock. Furthermore, expression of molecular clock genes will also fluctuate throughout the day in splenocytes, therefore, a reduction of clock gene expression due to PAMP challenge would be most significant if examined during the time of peak expression9. Since Dbp and Rev-erb&#945; have been shown to demonstrate significant expression peaks in splenocytes and splenic immune cells around the light-dark interphase 8,9,23,24 (ZT12), in the current method, cells were isolated and challenged at ZT13 in order to have a greater chance at detecting a reduction in these genes. Conversely, a PAMP that could increase clock expression, would most likely be observed if looking at a time of day when clock expression is at its lowest.
Since mice are nocturnal animals, their rest phase is during the light period, which corresponds to human activity. Therefore, ideally, mice will be housed in a room with minimal traffic and one that contains a white-noise machine in order to reduce daytime disturbances as this could disrupt the circadian rhythms of the mice. Furthermore, cage changes should be done well in advance of the experiment date. Any work in the animal room (including euthanasia of the animals) during the dark period, should be conducted under red light in order to avoid disrupting the rhythms of the mice.
Diurnal rhythms are subjected to environmental stimuli (e.g., light or food), which are termed zeitgebers. In the case of a 12 h light/12 h dark cycle, the zeitgeber (i.e., light) resets the clock to a 24 h period. While most diurnal rhythms are circadian (i.e., daily rhythms that occur in the absence of an external cue), they are not true circadian rhythms until they have been shown to oscillate with an approximate 24 h period under constant environmental conditions. Therefore, this procedure could be performed using mice under constant conditions, which would entail entraining mice to the light-dark cycle as described above, but then holding the animals in constant darkness for 3 days prior to sampling. This type of experiment is referred to as a dark-dark (DD) experiment and the time point of sampling would be referred to as CT (circadian time), not ZT.
While this method can identify PAMPs that alter clock gene expression within splenocytes, it does not take into account how these PAMPs affect the master clock or peripheral clocks throughout the body. Since the spleen is composed of a heterogeneous population of cells, individual PAMPs could impact each cell type differently. For example, Tlr9 expression rhythms in the mouse spleen differ between splenocytes, macrophages, B cells, and DCs15. Additionally, Tlr1, Tlr3, Tlr4, Tlr6, Tlr7,and Tlr8 displayed significant daily oscillations in an adherent splenocyte population but only Tlr2 and Tlr6 experience daily oscillations in enriched splenic macrophages24. Therefore, in order to investigate the outcome of a challenge on individual cell types, cells could be isolated via magnetic cell sorting, as previously described9,15 and then subsequently challenged. Additionally, the splenic cell population fluctuates over the daily cycle, which could also play a role in sensitivity to a particular PAMP and subsequent impact on the clock8.
This method allows for the isolation of a large number of immune cells that consist predominately of B cells (~58%), T cells (~21%), dendritic cells (~5%), and macrophages (~4%)25. The large number of cells provides the opportunity to challenge splenocytes with a variety of PAMPs in a single experiment. The splenocyte isolation procedure is very easy to perform, can be completed within minutes (depending on the number of animals), and with minimal animal or cellular manipulation, which is essential when examining the molecular clock because as mentioned above, these actions can disrupt the timing of the clock as well as clock-controlled genes. The results for this procedure were highly reproducible, as significance between challenged and unchallenged cells was achieved with just three animals and the results were consistent with previously published work23 (Figure 1). It should be noted that increasing the number of animals per group might have revealed statistically significant differences between a challenge group and control (e.g., ODN 1826 and Rev-erb&#945;).
Moving forward, while this protocol only addresses the acute effects on clock gene expression after PAMP challenge, it could provide proof of principle for further investigation. For example, this assay could be used as a model to decipher the molecular mechanisms regarding TLR - PAMP interaction and how it influences the molecular clock. It could also be used to determine the length of time it takes for the molecular clock to recover after a PAMP challenge, which could be determined by conducting a time-course experiment (i.e., assessing expression after varying times post challenge). As mentioned above, subsequent experiments could be performed to examine PAMP challenge on specific splenocyte cell populations. Since several pathogens stimulate multiple TLRs upon infection, it would be interesting to use this protocol to investigate if challenging with multiple PAMPs have a synergistic effect on clock gene expression.

The master clock in mammals, which orchestrates 24 h oscillations for nearly all aspects of physiology and behavior, is located within the suprachiasmatic nucleus (SCN) of the hypothalamus1,2. In addition to regulating biological processes on an organismal level, the master clock also synchronizes peripheral cellular clocks throughout the body3,4,5. While the molecular clock machinery consists of at least three interlocking transcriptional-translational feedback loops, the core is comprised of the Period (Per1-3), Cryptochrome (Cry1-2), Bmal1, and Clock genes6,7. Besides maintaining the accurate timing of the core molecular clock, some ancillary clock gene products (e.g., Rev-erb&#945; and Dbp) also regulate expression of non-clock genes, i.e., clock controlled genes (CCGs)6,7.
Functional molecular clocks have been described in various immune tissues (e.g., spleen and lymph nodes)8 and cells (e.g., B cells, dendritic cells, macrophages)8,9. These cells detect and respond to pathogen-associated molecular patterns (PAMPs), conserved microbial components, via innate immune recognition receptors such as Toll-like receptors (TLRs)10. To date, 13 functional TLRs have been described, which recognize microbial constituents such as bacterial cell wall components, flagellar protein, and microbial nucleic acids10. The PAMP, lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria recognized by TLR4, has been shown to alter circadian rhythms at the both organismal and molecular levels. For example, in vivo challenge of LPS induced photic-like phase delays as measured by activity in mice11 and led to reduced clock gene expression in the SCN and liver as determined by in situ hybridization and quantitative PCR, respectively, in rats12. After an in vivo challenge with LPS, analysis of human peripheral blood leukocytes13 and subcutaneous adipose tissue14 revealed altered expression of several clock genes as measured via qPCR. Lastly, ex vivo LPS challenges of human macrophages and mouse peritoneal macrophages, also led to altered clock expression as measured by qPCR14.
Here, we describe a protocol to assess the influence of the PAMPs LPS, ODN1826 (synthetic oligonucleotides containing unmethylated CpG motifs), and heat-killed Listeria monocytogenes (HKLM), recognized by TLR4, TLR9, and TLR2, respectively, on molecular clock gene expression in mouse splenocytes. The protocol includes mouse splenectomy, splenocyte isolation and challenge, RNA extraction, cDNA synthesis, and qPCR to assess expression of several clock genes. This protocol allows for the timely acquisition of a large number of immune cells with very little animal or cellular manipulation, which can then be challenged ex vivo with various PAMPs. The molecular clock has been shown to modulate various aspects of the immune response8,15,16, therefore, disruption of the molecular clock would most likely impair the proper time-dependent variation of the immune response. In addition, since disruptions of circadian rhythms can lead to serious pathologies17,18,19,20, it may be of interest for researchers to challenge splenocytes with a wide range of molecules and assess their influence on the clock.

<table><tbody><tr><td>Frosted slides</td><td>Fisher</td><td>12-550-343</td><td></td></tr><tr><td>Cell strainers</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Lipopolysaccharide&amp;nbsp;</td><td>InvivoGen</td><td>ltrl-eklps</td><td></td></tr><tr><td>ODN1826</td><td>InvivoGen</td><td>Tlrl-1826-1</td><td></td></tr><tr><td>HKLM</td><td>InvivoGen</td><td>Tlrl-hklm</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-043</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104 or 74106</td><td></td></tr><tr><td>RNase-Free DNase Set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>6-well cell culture plate</td><td>Denville</td><td>T1006</td><td></td></tr><tr><td>50 mL tubes</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>15 mL tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher</td><td>4368814</td><td></td></tr><tr><td>TaqMan Gene Expression Assays b-actin</td><td>ThermoFisher</td><td>Mm00607939_s1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Per2</td><td>ThermoFisher</td><td>Mm00478113_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Rev-erba</td><td>ThermoFisher</td><td>Mm00520708_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Bmal1</td><td>ThermoFisher</td><td>Mm00500226_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Dbp</td><td>ThermoFisher</td><td>Mm00497539_m1</td><td></td></tr><tr><td>qPCR machine StepOnePlus</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>ThermoFisher</td><td>4369016</td><td></td></tr><tr><td>MicroAmp Fast 96-well reaction plate (0.1 mL)</td><td>ThermoFisher</td><td>4346907</td><td></td></tr><tr><td>Statistical Analysis Software</td><td>Prism 7.0a</td><td></td><td></td></tr></tbody></table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.

Mice were sacrificed at ZT13, splenocytes were isolated and challenged ex vivo with the PAMPs LPS, ODN1826, or HKLM. After 3 h, RNA was isolated, and qPCR was used to assess relative expression levels of the molecular clock genes Clock, Per2, Dbp, and Rev-erb&#945; compared to unchallenged control cells. After PAMP challenge, Clock expression levels were not significantly different than expression in the control cells (Figure 1A). Per2 expression levels were significantly elevated in cells challenged with LPS and ODN1826 when compared to unchallenged controls (Figure 1B). LPS was the only PAMP to alter Rev-erb&#945; expression, as mRNA levels were significantly lower than in the unchallenged controls (Figure 1C). Lastly, significantly lower mRNA levels were observed for Dbp after challenge with each of the PAMPs when compared to the controls (Figure 1D). Consistent with what has been previously shown, out of all the clock associated genes examined, Dbp expression tends to be most affected by PAMP challenge23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58022/58022fig1.jpg" /> 
Figure 1: Altered clock gene expression in mouse splenocytes after ex vivo PAMP challenge. Splenocytes were isolated at ZT13 and challenged with LPS, ODN1826, or HKLM. Relative clock mRNA levels (normalized to &#946;-actin) were determined by qPCR 3 h after challenge. Each data point represents expression level for 1 animal. Experimental mean + SEM are given. *p &#60;0.05, **p &#60;0.01, ***p &#60;0.001. The indicated challenges were significantly different from the control (unchallenged cells) as per one-way ANOVA with the Dunnett&#39;s post hoc test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58022/58022fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

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Nanyang Technological University

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JoVE Journal

Genetics

7.4K Views.  University of Hartford. Dit protocol beschrijft een techniek met behulp van de muis splenocytes te ontdekken van de pathogeen-geassocieerde moleculaire patronen die veranderen van moleculaire klok genexpressie.

Video: The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Developmental Biology

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Immunology and Infection

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Neuroscience

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

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