Name: Video: Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy
Uploaded: 2016-11-08T13:00:00.000+00:00
Duration: 10 min
Description: 8.5K Views. Universidad Autónoma del Estado de Morelos. We beschrijven een standaard protocol antilichaam kleuring voor microscopie aan het membraan expressie en localisatie van gangliosiden in rustende en geactiveerde humane naïeve CD4 + T-cellen te bepalen. Ook beschreven zijn real-time PCR experimenten met

We are grateful to Dr. Jos&#233; Luis Daniotti for comments. We thank the assistance to Dr. J. Arturo Pimentel and the Laboratorio Nacional de Microscop&#236;a Avanzada-UNAM for acquisition of confocal images. I.M.-D. and T.M.V.C. were supported by grants 157634 and 253596 from Consejo Nacional de Ciencia y Tecnolog&#236;a (CONACYT), Sociedad Latinoamericana de Glicobiolog&#236;a, A.C. T.M.V.-C. is recipient of a scholarship (245192) from CONACYT. We also thank the support of the Red Tem&#225;tica Glicociencia en Salud - CONACYT (253596).

Peripheral blood from healthy male donors was obtained with informed consent and approval of the Bioethics Committee of the Centro de Investigaci&#242;n en Din&#225;mica Celular- Universidad Aut&#242;noma del Estado de Morelos.
1. Isolation and Activation of Human Na&#239;ve CD4+ T Cells
<ol>
	<li>Collect 2 ml of peripheral blood derived from healthy human donors through informed consent and dilute with 2 ml of sterile PBS-EDTA (1x Phosphate Buffered Saline (PBS), 2 mM EDTA, pH 7.4). Add the diluted blood slowly on top of 3 ml of sucrose solution with 1.077 density as previously described8. 
	Note: Differential migration during centrifugation caused by differences in density will cause erythrocytes to sediment completely and a layer of less dense mononuclear cells will form above. 2 ml of peripheral blood will render approximately 0.5 x 106 na&#239;ve CD4+ T cells after the purification steps.</li>
	<li>Centrifuge 30 min at 250 x g at 21 &#176;C (use rotor for round bottom tubes with fixed angle of 30 mm) with no break to generate the gradient of peripheral blood mononuclear cells (PBMCs). After centrifugation, the PBMCs can be clearly observed as a white ring. Collect the PBMCs with a pipette and transfer to sterile tube for washing.</li>
	<li>After three washes with PBS-EDTA solution at 250 x g for 10 min proceed to the purification of na&#239;ve CD4+ T cells. Sorting methods or several types of purification kits are available for this purpose. Preferably, use &#62;1 x 107 PBMCs for purification.
	<ol>
		<li>Purify na&#239;ve CD4+ T cells by negative selection with magnetic beads. Perform negative selection with anti CD45RO, CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD56, CD123, anti-TCR&#947;/&#948;, anti-HLA-DR, and CD235a (glycophorin A) antibodies. Assess the percentage of purity by determining CD4+CD45RA+ cells through flow cytometry. 
		Note: Optionally, proceed to incubation of PBMCs overnight at 37 &#176;C and 5 % CO2 with a density of 1 x 106 cells/ml in 10 ml of supplemented medium to promote monocyte adherence and continue with na&#239;ve CD4+ T cell purification. The efficient recovery of na&#239;ve CD4+ T cells from peripheral blood depends largely on the purification system.</li>
	</ol>
	</li>
	<li>Prepare 24-well cell culture plates by adding 250 &#181;l per well of PBS with 5 &#181;g/ml anti CD3 monoclonal antibody and incubate for at least 2 hr at 37 &#176;C. 
	Note: Plates with 96 wells can be used when a smaller number from na&#239;ve CD4+ T cells are used (e.g., 2 x104-1 x 105).</li>
	<li>Remove the anti CD3 antibody dilution from the 24 well or 96 well plates and wash the plates twice using sterile 1x PBS.</li>
	<li>Dilute the na&#239;ve CD4+ T cells with &#62;95 % of purity (CD4+CD45RA+) to a density of 1 x 106 cells/ml in advanced RPMI 1640 medium supplemented with 3 % of fetal bovine serum (or RPMI supplemented with 10 % of serum), 2 mM glutamine and antibiotics (1 U/ml penicillin, 1 &#181;g/ml streptomycin). 
	Note: If ganglioside localization is required in PBMCs, follow the same protocol for dilution and stimulation as described below.</li>
	<li>Add 500 &#181;l of the cell dilution to anti CD3 coated wells and to non-coated wells (control cells) in the 24 well plate. Alternatively, add 100 &#181;l of the cell dilution to the wells of the 96 well plate.</li>
	<li>Complete the stimulation conditions of na&#239;ve CD4+ T cells in anti CD3 coated wells by adding 1 &#181;g/ml of anti CD28 antibody.</li>
	<li>Keep the resting CD4+ T cells as control in the non-coated wells without addition of anti CD28 antibodies. Incubate the resting and activated CD4+ T cell for 0 to 72 hr under standard conditions of 37 &#176;C and 5% CO2.</li>
	<li>To verify an adequate activation evaluate by flow cytometry the expression of the CD69 early activation marker (16 hr post-activation) or CD25 late activation marker (48 hr post-activation) in resting and activated na&#239;ve CD4+ T cells incubated at 37 &#176;C and 5% CO2 in absence or presence of anti CD3/CD28 antibodies9,10.</li>
</ol>
2. Adherence of Resting and Activated Na&#239;ve CD4+ T Cells
<ol>
	<li>Sterilize 12 mm round coverslips by autoclaving and UV light exposure for at least 15 min.</li>
	<li>Place coverslips in sterile 24-well cell culture plates. 
	Note: For cultures with less than 1 x 105 cells it is preferable to use slide chambers with adapted small wells to prevent dispersion of cells in the coverslip.</li>
	<li>Coat coverslips (or slide chamber) with 200 &#181;l of poly-L-lysine (M.W. 150-300 KDa) 0.1% (w/v) solution and incubate for 5 min at room temperature (RT), remove and dry for at least 1 hr at RT.</li>
	<li>Mix carefully and collect the resting and activated na&#239;ve CD4+ T cells from the 24-well plates. Count the cells and if necessary adjust with fresh supplemented medium to transfer 1 x 105 cells to the poly-L-lysine coated coverslips. Incubate the cells for a minimum of 6 hr at 37 oC and 5% CO2.</li>
</ol>
3. Collecting and Fixation of Resting and Activated Na&#239;ve CD4+ T Cells
<ol>
	<li>Carefully remove the medium from cultured resting and activated na&#239;ve CD4+ T cells.</li>
	<li>Add 500 &#181;l of RT sterile PBS slowly. 
	Note: Careful addition and removal of solutions from wells prevents the detachment of cells from the coverslip.</li>
	<li>Discard the PBS and add another 500 &#181;l PBS to thoroughly remove culture media.</li>
	<li>Fix the cells by addition of 500 &#181;l filtered 4% paraformaldehyde (filtration removes microparticles that can interfere with microscopy analysis) and incubate for 30 min at RT (preferred) or overnight at 4 &#176;C. 
	Caution: Paraformaldehyde is a toxic and volatile compound. It is known to be a human carcinogen. Use carefully with an appropriate safety protocol.</li>
	<li>Remove the fixative and wash at least twice with PBS at RT.</li>
	<li>Proceed to the next step or maintain the plate wells with 500 &#181;l of PBS at 4 &#176;C for several days until staining. 
	Note: Sterility during this process helps avoid microorganism growth.</li>
</ol>
4. Staining, Mounting and Visualization by Confocal Microscopy
<ol>
	<li>Remove the PBS from the wells. Add 500 &#181;l of cold blocking buffer (1x PBS, 0.5% standard grade bovine serum albumin, 1% fetal bovine serum pH 7.4). Incubate for 20 min on ice.</li>
	<li>Remove the blocking buffer carefully and add the primary antibodies (anti gangliosides GD3 clone R24, GD2 clone 14G2a), PE-conjugated anti CD25 and APC-conjugated anti TCR and isotypes controls diluted in blocking buffer to 2.5 &#181;g/ml.</li>
	<li>Incubate 2 hr on ice or overnight at 4 &#176;C. 
	Note: Slow orbital agitation is preferable.</li>
	<li>Remove the antibody carefully and add slowly 500 &#181;l of PBS. Repeat twice.</li>
	<li>Add the secondary antibodies (FITC conjugated anti IgG3 for R24 anti GD3, Alexa Fluor 488-conjugated or Alexa Fluor 647-conjugated anti IgG2a for 14G2a anti GD2) diluted in blocking buffer to 0.1 &#181;g/ml.</li>
	<li>Incubate for 1 hr on ice in the dark without shaking.</li>
	<li>Wash three times in PBS as described in 4.4). Keep the wells with enough PBS to avoid the dehydration of samples.</li>
	<li>Add Hoechst 333258 stain diluted in PBS to 0.1 &#181;g/ml.</li>
	<li>Incubate 15 min at RT and remove. Wash three times in PBS.</li>
	<li>Place the slides on a paper towel and add 20 &#181;l of mounting solution (50% glycerol in PBS) or commercial mounting solutions to avoid fading and quenching from samples.</li>
	<li>Carefully pick up the coverslip with fine tweezers and place it on the mounting solution. Ensure that the side of the coverslip where cells are attached is in contact with the solution. Seal the coverslips with nail polish and analyze using fluorescence or confocal microscopy.</li>
</ol>
5. Isolation of RNA
<ol>
	<li>Prepare anti CD3 antibody (5 &#181;g/ml) coated 96-well plates. Incubate 4 x 104 na&#239;ve CD4+ T cells/well in 100 &#181;l of supplemented culture medium with anti CD28 antibody (1 &#181;g/ml) to complete the stimulus. Incubate at 37 &#176;C and 5% CO2 for 0 to 72 hr.</li>
	<li>After different post-activation times place the cells into a microcentrifuge tube.</li>
	<li>Add 500 &#181;l of PBS and centrifuge at 250 x g for 5 min at RT.</li>
	<li>Remove the supernatant and add 1ml Trizol reagent.</li>
	<li>Incubate for 5 min at RT and keep the sample at -80 &#176;C for at least 24 hr. Proceed with the RNA isolation when needed. 
	Note: Leaving the sample at -80 &#176;C for at least 24 hr improves RNA yield. Also, use of hand gloves is essential to avoid RNA degradation by RNases.</li>
	<li>Defrost the sample by incubation at 37 &#176;C for 2 min in a water bath.</li>
	<li>Add 200 &#181;l chloroform and shake vigorously by hand for 30 sec (to avoid aggressive mixing of RNA by vortexing). Incubate 5 min at RT.</li>
	<li>Centrifuge 15 min at 12,000 x g at 4 &#176;C.</li>
	<li>Recover the aqueous phase into a new microcentrifuge tube.</li>
	<li>Add 500 &#181;l isopropanol and invert the tube three times.</li>
	<li>Incubate 10 min at RT and centrifuge for 10 min at 12,000 x g at 4 &#176;C.</li>
	<li>Discard the supernatant by inversion.</li>
	<li>Add 1 ml of ice cold 75% ethanol and centrifuge 10 min at 12,000 x g at 4 &#176;C.</li>
	<li>Completely discard the supernatant and dry the RNA pellet by leaving the cap of the tube open. 
	Note: At this point the pellet is not clearly visible. Proceed anyway.</li>
	<li>Resuspend the pellet in 20 &#181;l of molecular grade water. Calculate the concentration and purity of nucleic acids by measuring the 260 nm and 280 nm absorbance using nanodrop.</li>
	<li>Proceed carefully to synthesis of cDNA by using any conventional reverse transcriptase kit and following the manufacturer&#180;s protocol. 
	Note: Approximately 0.5-2 &#181;g RNA is obtained from 4 x 104 na&#239;ve CD4+ T cells. cDNA can be synthetized from 100 ng of RNA and used in real-time PCR reactions without the need for low RNA input kits.</li>
</ol>

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The authors have nothing to disclose.

The described protocol can be used to localize gangliosides or proteins in cell suspensions of CD4+ T cells or other immune cells (e.g., PMBCs, Figure 5) starting from a small number of cells. Because of the small size of T cells and non-adherent properties, the acquisition of fluorescence microscopic images results in poor information or low quality if the cells are not correctly adhered.
This protocol combined with a good quality confocal microscopy analysis is a key advantage over the use of techniques such as thin layer chromatography for detection of gangliosides because it reveals the spatial localization and dynamics of gangliosides during treatment (e.g., T cell activation), leading to acquisition of biologically relevant data11,12,13. Also, this protocol allows the evaluation of ganglioside expression starting from a small number of CD4+ T cells (2 x 104) that is useful for optimizing the number of replicates or different assays6.
The careful treatment during washes and antibody staining after adhesion is crucial to maintain cell number and integrity. Because of the weak binding of CD4+ T cells to the coverslip, it is also very important to carefully add and remove solutions; otherwise a reduction in cell number and increased fluorescent debris will be encountered. When assessed through confocal microscopy, the immunostaining of gangliosides confers additional information such as spatial localization or co-localization with other molecules in the plasma membrane or intracellular compartments. Subcellular localization requires the use of subcellular markers14,15. In addition, fresh and not fixed CD4+ T cells need to be used for staining when determining ganglioside expression restricted to the cell surface. Although fixation of CD4+ T cells helps preserve the sample for at least 5 days, it carries the risk of permeabilization and intracellular staining.
However, because of the structural similarity between gangliosides, it is always advisable to take into account the specificity of antibodies used. Several of the commercially available anti-ganglioside monoclonal antibodies can be used and for some of them the specificity has been probed by ELISA, TLC, etc.16,17,18.
Previous works demonstrate that it is possible to obtain information regarding localization and expression of GD3 and GD2 gangliosides from 2 x 104 cells/per condition6. Also, this protocol describes the capability of performing gene profiling experiments from 4 x 104 cells, allowing optimization of silencing experiments such as those based in lentiviral particles6.

The CD4+ T cells orchestrate the immune response through their effector functions after activation by antigen presenting cells1. The study of the cellular mechanisms that are modulated during activation allows insight into a basic process of immune function. However, the study of na&#239;ve CD4+ T cells can be complicated because they represent a very small population of cells in the blood periphery2.
Through fluorescence microscopy several reports have studied the localization of different molecules involved in CD4+ T cell activation, mainly proteins associated to the plasma membrane3. The gangliosides are sialic acid containing glycosphingolipids and although they have been extensively studied in nerve cells where they are abundant, other cells such as immune cells also express gangliosides with biologically relevant functions4,5. We previously reported that during activation of human na&#239;ve CD4+ T cells there is an upregulation of the &#945;2,8 sialyltransferase ST8Sia 1 (GD3 synthase) and the GM2/GD2 synthase, that induce the significant surface neoexpression of GD2 and the upregulation of GD3 ganglioside6. Further study of GD3, GD2 and other gangliosides in immune cells is necessary to complement a protein-based partial view of immune function.
Commonly, the study of ganglioside expression is based on techniques such as Thin Layer Chromatography (TLC)7, but this technique does not allow the spatial localization of gangliosides at the plasma membrane or in subcellular compartments, limiting biological analysis.
In this work, we describe a protocol for the antibody-mediated identification and localization of GD3 and GD2 gangliosides in human na&#239;ve CD4+ T cells and PBMCs after anti-CD3/anti-CD28 activation. With this protocol it is also possible to analyze the gene and molecular expression of gangliosides in a low number of cells in suspension, with acquisition of high quality images6, considering the small size of lymphocytes (9 &#181;m).

<table><tbody><tr><td>Advanced RPMI 1640&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>12633-012</td><td>Suplemented with 3% FBS</td></tr><tr><td>mouse anti human CD3 antibody</td><td>eBioscience</td><td>16-0037-81</td><td>OKT3 clone</td></tr><tr><td>mouse anti human CD28 antibody</td><td>ebioscience</td><td>16-0288-81</td><td>CD28.6 clone</td></tr><tr><td>mouse anti human GD3&amp;nbsp; antibody</td><td>Abcam</td><td>ab11779</td><td>R24 clone</td></tr><tr><td>mouse anti human GD2 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-53831</td><td>14G2a clone</td></tr><tr><td>FITC-conjugated anti-mouse IgG3&lt;br/&gt; antibody</td><td>Abcam</td><td>ab97259</td><td></td></tr><tr><td>Alexa Fluor 488 conjugated antimouse</td><td>Thermo Fisher Scieni&amp;iexcl;tific</td><td>A-21131</td><td></td></tr><tr><td>IgG2a antibody</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 647 conjugated antimouse</td><td>Thermo Fisher Scieni&amp;iexcl;tific</td><td>A-21241</td><td></td></tr><tr><td>IgG2a antibody</td><td></td><td></td><td></td></tr><tr><td>Hoechst 333258</td><td>Sigma-Aldrich</td><td>861405</td><td></td></tr><tr><td>&amp;nbsp;Ficoll Paque-Plus</td><td>GE</td><td>17-1440-02&amp;nbsp;</td><td></td></tr><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596-026</td><td></td></tr><tr><td>Naive CD4+ T Cell Isolation Kit II, human</td><td>MACS Miltenyi Biotec</td><td>130-094-131</td><td></td></tr><tr><td>BD FACSAria II</td><td>BD Biosciences</td><td></td><td>Sorting&amp;nbsp;</td></tr><tr><td>Nunc Lab-tek chamber slide system</td><td>Sigma-Aldrich</td><td>C7182</td><td></td></tr><tr><td>Olympus FV 1000 Laser Confocal Microscope (Olympus)&amp;nbsp;</td><td>Olympus</td><td></td><td>Upright BX61WI and IX81&amp;nbsp;</td></tr><tr><td>Forward sequence primer GD3 synthase</td><td>5&amp;acute;-GAGCGTTCAGGAAACAAATGG- 3&amp;acute;</td><td></td><td>Ref. 7</td></tr><tr><td>Reverse sequence primer GD3 synthase</td><td>5&amp;acute;-CCTGTGGGAAGAGAGAGTAAG-3&amp;acute;</td><td></td><td>Ref. 7</td></tr><tr><td>Forward sequence primer GM2/GD2 synthase</td><td>5&amp;acute;-CAACACAGCAGACACAGTCC-3&amp;acute;</td><td></td><td>Ref. 7</td></tr><tr><td>Reverse sequence primer GM2/GD2 synthase</td><td>5&amp;acute;-GTGGCAATCGTGACTAGAGC-3&amp;acute;</td><td></td><td>Ref. 7</td></tr></tbody></table>

preparation of cd4+ t cells for analysis of gd3 and gd2 ganglioside membrane expression by microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

The protocol described in this manuscript renders a good quality of cultured and adhered resting and activated human na&#239;ve CD4+ T cells (Figure 1). The activated CD4+ T cells show the characteristic proliferative profile (Figure 1B) in comparison to the resting condition (Figure 1A). The CD25 late activation marker is useful to evaluate the efficient activation at 72 hr observed by confocal microscopy (Figure 1C). The CD69 marker is currently used as early activation marker by flow cytometry or microscopy. Adherence of resting and activated na&#239;ve CD4+ T cells to a coverslip coated with poly-L-lysine are useful to study the expression and localization of GD3 and TCR showed by a double labeling with anti GD3 and anti TCR antibodies (Figure 2). As we reported previously, activation is accompanied by remodeling of GD3 and GD2 gangliosides in the cells surface6. Additionally, gene expression quantification of the GD3 and GM2/GD2 synthases is performed from 4 x 104 cells (Figures 2 and 3). The neo-expression of the GD2 ganglioside and TCR colocalization is adequately observed by confocal microscopy, demonstrating a possible role of GD2 in TCR clustering during activation (Figure 3). Figure 4 shows the localization of the GD3 and GD2 ganglioside stained simultaneously in activated CD4+ T cells. Additionally, activated PBMCs were stained with anti GD3 and anti GD2 antibodies showing that this protocol can be used in other immune cell populations found in PMBCs, particularly GD2 that was found to be expressed in all the activated PMBCs (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/54569/54569fig1.jpg" /> 
Figure 1: Visualization of na&#239;ve CD4+ T cells after adherence and efficient anti CD3/anti-CD28 activation. (A) Resting CD4+ T cells at 72 hr post-activation attached on poly-L-lysine treated coverslip are observed by brightfield microscopy. (B) Activated CD4+ T cells at 72 hr attached on poly-L-lysine treated coverslip. (C) CD25 marker expression (red) and Hoechst 333258 stained nuclei (blue) of anti CD3/anti CD28 activated CD4+ T cells at 72 hr post-activation. Scale bar = 50 &#181;m. Images were obtained by confocal microscopy with a 60X S/1.3 oil objective with 2X digital zoom. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54569/54569fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54569/54569fig2.jpg" /> 
Figure 2: GD3 ganglioside and TCR stain and microscopy localization in resting and activated na&#239;ve CD4+ T cells at 72 h post-activation. Resting CD4+ T cells with (A) nuclei stained with Hoechst 333258, (B) GD3 ganglioside localization, (C) Merge of nuclei and GD3 ganglioside. Activated CD4+ T cells with (D) nuclei stained with Hoechst 333258, (E) GD3 ganglioside localization and (F) Merge of nuclei and GD3 ganglioside. (G) and (H) show TCR and GD3 ganglioside in resting na&#239;ve CD4+ T cells. (J and K) corresponds to TCR and GD3 ganglioside staining in activated CD4+ T cells. Confocal images of TCR (red) and GD3 ganglioside (green) localization in resting na&#239;ve CD4+ T cells (I) and anti CD3/CD28 Activated na&#239;ve CD4+ T cells 72 hr post-activation (L). The GD3, TCR and nuclei stain was assessed by confocal microscopy from one stack with a 60X S/1.3 oil objective with 2X digital zoom. (M) The graph shows the gene expression for GD3 synthase determined by real-time PCR expressed as Fold change from 4 x 104 resting (Rest) and activated (Act) na&#239;ve CD4+ T cells at 72 hr post-activation. Data are the mean &#177; S. D. from three independent donors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54569/54569fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54569/54569fig3.jpg" /> 
Figure 3: GD2 ganglioside and TCR stain and microscopy localization in resting and activated na&#239;ve CD4+T cells at 72 h post-activation. Resting CD4+ T cells with (A) nuclei stained with Hoechst 333258, (B) GD2 ganglioside localization, (C) Merge of nuclei and GD2 ganglioside. Activated CD4+ T cells with (D) nuclei stained with Hoechst 333258, (E) GD2 ganglioside localization and (F) Merge of nuclei and GD2 ganglioside. (G and H) show TCR and GD2 ganglioside in resting na&#239;ve CD4+ T cells. (J and K) corresponds to TCR and GD2 ganglioside staining in activated CD4+ T cells. Confocal images of TCR (red) and GD2 ganglioside (green) localization in resting na&#239;ve CD4+ T cells (I) and anti CD3/CD28 Activated na&#239;ve CD4+ T cells 72 hr post-activation (L). The GD2, TCR and nuclei stain was assessed by confocal microscopy from one stack with a 60X S/1.3 oil objective with 2X digital zoom. (M) The graph shows the gene expression for GM2/GD2 synthase determined by real-time PCR expressed as Fold change from 4 x 104 resting (Rest) and activated (Act) na&#239;ve CD4+ T cells at 72 hr post-activation. Data are the mean &#177; S. D. from three independent donors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54569/54569fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54569/54569fig4.jpg" /> 
Figure 4: Double stain with anti-GD3 and anti-GD2 antibodies for GD3 and GD2 gangliosides localization. (A) GD3 ganglioside identified with R24 anti GD3 antibody in activated CD4+ T cells shows intracellular and membrane localization. (B) GD2 ganglioside identified with 14G2a anti GD2 antibody in activated CD4+ T cells shows only plasma membrane localization. (C) Brightfield microscopy from activated CD4+ T cells. The GD3 and GD2 stain was assessed by confocal microscopy from one stack with a 60X S/1.3 oil objective with 2X digital zoom. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54569/54569fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54569/54569fig5.jpg" /> 
Figure 5: GD3 and GD2 ganglioside localization in activated PBMCs. (A) GD2 ganglioside was detected with 14G2a anti GD2 antibody in anti CD3/anti CD28 72 hr post-activated PBMCs overlay with brightfield microscopy, (B) GD2 ganglioside expression and (C) GD2 ganglioside and nuclei merge. (D) GD3 ganglioside stained with R24 anti GD3 antibody overlay with brightfield, (E) GD3 ganglioside expression and (F) GD3 ganglioside and nuclei merge. The GD3, GD2 and nuclei stain was assessed by confocal microscopy from one stack with a 60X S/1.3 oil objective with 2X digital zoom. Scale bar = 45 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54569/54569fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy at JoVE.com

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

preparation-cd4-t-cells-for-analysis-gd3-gd2-ganglioside-membrane

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.5K Views.  Universidad Autónoma del Estado de Morelos.  We beschrijven een standaard protocol antilichaam kleuring voor microscopie aan het membraan expressie en localisatie van gangliosiden in rustende en geactiveerde humane naïeve CD4 + T-cellen te bepalen. Ook beschreven zijn real-time PCR experimenten met 

Video: Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

Biology

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Biochemistry

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

Neuroscience

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

An In Vitro Technique to Differentiate GM-CSF Producing T Helper Cells from Na&iuml;ve T Cells

Source: Lu, Y., et al.&#160;<a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58087">In Vitro&#160;Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates the in vitro differentiation of granulocyte-macrophage-colony-stimulating factor or GM-CSF-producing T helper cells (THGM). Isolated na&#239;ve T cells are stimulated for differentiation into THGM, which is confirmed by detecting high intracellular GM-CSF production and a low expression of other T helper cell-specific cytokines.

Source: Lu, Y., et al.&#160;In Vitro&#160;Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Differentiation Techniques

A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds

Source: Fouda, A., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55199">A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules.</a> J. Vis. Exp., (2017).
This video demonstrates the screening of immunomodulatory compounds via fluorescent-based assay. Transgenic mouse-derived T lymphocytes with green fluorescent protein expression cassettes are treated with these compounds. The enhanced and reduced GFP signals enable the identification of the compounds&#39; stimulatory and inhibitory effects, respectively.

1. Preparation of Splenocyte Medium and Flow-cytometry Buffer

	Perform all steps under a 70% ethanol-cleaned biological hood.
	Remove 70 mL of pre-warmed ...

Preparation of CD4⁺ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Samenvatting

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