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....

<ol>
	<li>Mazzon, C., Viola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+tango+to+quadrilla:+current+views+of+the+immunological+synapse.">From tango to quadrilla: current views of the immunological synapse.</a> Cell Adh Migr. 1 (1), 7-12 (2007).</li><li>Hamann, D., Pa Baars, ., Hooibrink, B., van Lier, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+the+human+CD4++T-cell+population:+two+distinct+CD4++T-cell+subsets+characterized+by+coexpression+of+CD45RA+and+CD45RO+isoforms.">Heterogeneity of the human CD4+ T-cell population: two distinct CD4+ T-cell subsets characterized by coexpression of CD45RA and CD45RO isoforms.</a> Blood. 88 (9), 3513-3521 (1996).</li><li>Zhong, L., Zhang, Z., Lu, X., Liu, S., Chen, C. Y., Chen, Z. 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C., Barral, A., Barral Netto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+human+peripheral+blood+monocyte+isolation.">A simple method for human peripheral blood monocyte isolation.</a> Mem Inst Oswaldo Cruz. 95 (2), 221-223 (2000).</li><li>O'Neil-Andersen, N. J., Lawrence, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+modulation+of+surface+and+intracellular+protein+expression+by+T+cells+after+stimulation+in+the+presence+of+monensin+or+brefeldin+A.">Differential modulation of surface and intracellular protein expression by T cells after stimulation in the presence of monensin or brefeldin A.</a> Clin Diagn Lab Immunol. 9 (2), 243-250 (2002).</li><li>Mannering, S. I., Zhong, J., Cheers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+activation,+proliferation+and+apoptosis+in+primary+Listeria+monocytogenes+infection.">T-cell activation, proliferation and apoptosis in primary Listeria monocytogenes infection.</a> Immunology. 106 (1), 87-95 (2002).</li><li>Marconi, S., Acler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-GD2-like+IgM+autoreactivity+in+multiple+sclerosis+patients.">Anti-GD2-like IgM autoreactivity in multiple sclerosis patients.</a> Mult Scler. 12 (3), 302-308 (2006).</li><li>Park, J. E., Wu, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+specificity+of+natural+killer+T+cells+against+GD3+ganglioside+and+identification+of+GM3+as+an+inhibitory+natural+killer+T-cell+ligand.">Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand.</a> Immunology. 123 (1), 145-155 (2008).</li><li>Simon, B. M., Malisan, F., Testi, R., Nicotera, P., Leist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disialoganglioside+GD3+is+released+by+microglia+and+induces+oligodendrocyte+apoptosis.">Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis.</a> Cell Death Differ. 9 (7), 758-767 (2002).</li><li>Beske, O., Reichelt, M., Taylor, M. P., Kirkegaard, K., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poliovirus+infection+blocks+ERGIC-to-Golgi+trafficking+and+induces+microtubule-dependent+disruption+of+the+Golgi+complex.">Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex.</a> J Cell Sci. 120 (18), 3207-3218 (2007).</li><li>Zuber, C., Spiro, M. J., Guhl, B., Spiro, R. G., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golgi+apparatus+immunolocalization+of+endomannosidase+suggests+post-endoplasmic+reticulum+glucose+trimming:+implications+for+quality+control.">Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control.</a> Mol Biol Cell. 11 (12), 4227-4240 (2000).</li><li>Yamashiro, S., Okada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+(GD3+synthase)+gene+in+human+cancer+cell+lines:+high+level+expression+in+melanomas+and+up-regulation+in+activated+T+lymphocytes.">Expression of (GD3 synthase) gene in human cancer cell lines: high level expression in melanomas and up-regulation in activated T lymphocytes.</a> Glycoconj J. 12 (6), 894-900 (1995).</li><li>Wipfler, D., Srinivasan, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentially+regulated+expression+of+9-O-acetyl+GD3+(CD60b)+and+7-O-acetyl-GD3+(CD60c)+during+differentiation+and+maturation+of+human+T+and+B+lymphocytes.">Differentially regulated expression of 9-O-acetyl GD3 (CD60b) and 7-O-acetyl-GD3 (CD60c) during differentiation and maturation of human T and B lymphocytes.</a> Glycobiology. 21 (9), 1161-1172 (2011).</li><li>Pukel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., Old, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GD3,+a+prominent+ganglioside+of+human+melanoma.+Detection+and+characterisation+by+mouse+monoclonal+antibody.">GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody.</a> J Exp Med. 155 (4), 1133-1147 (1982).</li></ol>

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 analy...

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 border="0" cellpadding="0" cellspacing="0" width="1052">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Advanced RPMI 1640&#160;</td>
			<td style="width:241px;">Thermo Fisher Scientific</td>
			<td style="width:137px;">12633-012</td>
			<td style="width:193px;">Suplemented with 3% FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD3 antibody</td>
			<td>eBioscience</td>
			<td>16-0037-81</td>
			<td>OKT3 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD28 antibody</td>
			<td>ebioscience</td>
			<td>16-0288-81</td>
			<td>CD28.6 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD3&#160; antibody</td>
			<td>Abcam</td>
			<td>ab11779</td>
			<td>R24 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD2 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53831</td>
			<td>14G2a clone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:481px;">FITC-conjugated anti-mouse IgG3 
			antibody</td>
			<td>Abcam</td>
			<td>ab97259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 333258</td>
			<td>Sigma-Aldrich</td>
			<td>861405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Ficoll Paque-Plus</td>
			<td>GE</td>
			<td>17-1440-02&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Naive CD4+ T Cell Isolation Kit II, human</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-094-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSAria II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Sorting&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab-tek chamber slide system</td>
			<td>Sigma-Aldrich</td>
			<td>C7182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus FV 1000 Laser Confocal Microscope (Olympus)&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Upright BX61WI and IX81&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GD3 synthase</td>
			<td>5&#180;-GAGCGTTCAGGAAACAAATGG- 3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GD3 synthase</td>
			<td>5&#180;-CCTGTGGGAAGAGAGAGTAAG-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-CAACACAGCAGACACAGTCC-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-GTGGCAATCGTGACTAGAGC-3&#180;</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 (F...

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 ...

The overall goal of this protocol is to demonstrate the preparation of a CD4+T cell suspension for staining to visualize GD3 and GD2 ganglioside localization on the cell surface using a low number of cells. This method visualization of membrane glycolipids in a low number of small suspension cells, as is the case of mononuclear cells. The main advantage of this technique is that you can evaluate ganglioside expression and localization in low amounts of CD4+T cells by optimizing sample use.The implications of this technique could extend toward research and diagnosis of some types of T cell leukemia. As we have found, some leukemia cell lines upregulate GD3 and GD2 ganglioside expression. Begin by diluting two milliliters of peripheral blood from healthy human donors with two milliliters of sterile PBS EDTA.Slowly dispense the diluted blood on top of three milliliters of 1.077 density sucrose solution. Then, centrifuge for 30 minutes at 250 times G at 21 degrees Celsius with no break to generate the gradient of peripheral blood mononuclear cells. After centrifugateion the PBMCs can be clearly observed as a white ring.Collect the PBMCs with a pipette and transfer to a sterile tube for washing. After washing the cells with PBS EDTA, purify naive CD4+T cells by negative selection with magnetic beads using a commercially available kit. Once a population of greater than 95%purity CD4+T cells has been obtained, prepare 24 well cell culture plates by adding 250 microliters of five micrograms per milliliter anti-CD3 monoclonal antibody in PBS to each well.Incubate the plate at 37 degrees Celsius for at least two hours. Following the incubation, remove the anti-CD3 antibody dilution from the plates and wash twice using sterile 1x PBS. Then, dilute the naive CD4+T cells with supplemented advanced RPMI 1640 medium to a density of one times 10 to the sixth cells per milliliter.Next, add 500 microliters of diluted cells to the anti-CD3 coated wells of the 24 well plate. Then complete the stimulation of naive CD4+T cells in anti-CD3 coated wells by adding one microgram per milliliter anti-CD28 antibody. Incubate the resting and activated CD4+T cells for zero to 72 hours at 37 degrees Celsius and 5%carbon dioxide.After the allotted incubation time has elapsed, collect the resting and activated naive CD4+T cells from the 24 well plates. Perform a cell count and if necessary, dilute the cells with fresh supplemented medium to a concentration of one times 10 to the sixth cells per milliliter. Seed 500 microliters of diluted cells into the wells of a 24 well plate containing poly-L-lysine coated cover slips or a slide chamber and incubate at 37 degrees Celsius and 5%carbon dioxide for a minimum of six hours.Following the incubation, the CD4+T cells will be adhered to the cover slips. Carefully remove the medium from the wells and then slowly add 500 microliters of sterile PBS. PBS addition during washing should be done very carefully, especially with slide chambers with less than 50, 000 cells per well are being prepared.Aspirate the PBS and add another 500 microliters of PBS to the wells to thoroughly remove the culture medium. After removing all of the PBS, add 500 microliters of filtered 4%paraformaldehyde and incubate for 30 minutes at room temperature to fix the cells. Following fixation, remove the fixative and wash at least twice with PBS.If desired, the plate can be stored with 500 microliters of PBS in each well for several days at four degrees Celsius. First, remove the PBS from the fixed cells and add 500 microliters of cold blocking buffer. Incubate for 20 minutes on ice.During incubation, it is critical not to vigorously shake the plate, as shaking will take off the cells. Carefully aspirate the blocking buffer and add blocking buffer containing 2.5 micrograms per milliliter of one of the primary antibodies or PE conjugated anti-CD25, APC conjugated anti-TCR, or an isotype control. Incubate for two hours on ice and protect from light or overnight at four degrees Celsius.Following the incubation, carefully remove the antibody solution and slowly add 500 microliters of PBS. Then add the appropriate secondary antibodies diluted in blocking buffer to 0.1 micrograms per milliliter. Protect from light and incubate for one hour on ice while taking care to avoid shaking.After washing three times in PBS as before, add Hoecht stain diluted in PBS to 0.1 micrograms per milliliter and incubate protected from light for 15 minutes at room temperature. Then place the washed slides on a paper towel and add 20 microliters of mounting solution or commercial mounting solution to prevent fading and quenching of fluorescence. Finally, pick up a cover slip with fine tweezers, turn it face down and place it on top of the mounting solution so that the side of the cover slip where cells are attached is in contact with the solution.Seal the cover slips with nail polish and analyze using fluorescence or confocal microscopy. This image shows bright field visualization of fixed CD4+T cells 72 hours after activation. Validation of efficient activation after staining is assessed using anti-CD25 antibody shown in red.The GD3 ganglioside is localized intracellularly and on the cell surface of activated CD4+T cells, as assessed by staining with anti-GD3 antibody shown in green. GD3, again shown in green, does not co-localize with the TCR, shown again in red, as assessed with anti-GD3 and anti-TCR antibody staining. However, the GD2 ganglioside does co-localize with the TCR and CD4+T cells, as assessed by staining with anti-GD2, shown in green, and anti-TCR antibody staining, shown in red.The GD3 ganglioside staining shows the intracellular and cell surface localization in a subset of activated PBMCs. While the GD2 ganglioside staining shows intracellular and cell surface localization in all activated PBMCs. Following this procedure of adherence and manipulation of the CD4+T cells, other methods like intracellular compartment staining can be performed in order to answer additional questions like the specific intracellular localization or processing of gangliosides.After watching this video, you should have a good understanding of how to handle and adhere a low number of CD4+T cells to obtain high quality fluorescent microscopy images to analyze ganglioside expression.

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

Research

JoVE Journal

Immunology and Infection

8.4K 조회수.  Universidad Autónoma del Estado de Morelos. The overall goal of this protocol is to demonstrate the preparation of a CD4+T cell suspension for staining to visualize GD3 and GD2 ganglioside localization on the cell surface using a low number of cells. This method visualization of membrane glycolipids in a low number of small suspension cells, as is the case of mononuclear cells. The main advantage of this technique is that you can evaluate ganglioside expression and localization in low amounts of CD4+T cells by optimizing sample use.