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 naïve CD4+ T cells. Also described are real-time PCR experiments using <40,000 cells that do not require additional low input RNA kits.
The methods described herein for activation of naïve 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 naïve CD4+ T cell activation. Also, real-time PCR experiments using <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 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 naïve 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 naïve CD4+ T cells there is an upregulation of the α2,8 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 naïve 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 µm).
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 authors have nothing to disclose.
We are grateful to Dr. José Luis Daniotti for comments. We thank the assistance to Dr. J. Arturo Pimentel and the Laboratorio Nacional de Microscopìa 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 Tecnologìa (CONACYT), Sociedad Latinoamericana de Glicobiologìa, A.C. T.M.V.-C. is recipient of a scholarship (245192) from CONACYT. We also thank the support of the Red Temática Glicociencia en Salud – CONACYT (253596).
Advanced RPMI 1640 | Thermo Fisher Scientific | 12633-012 | Suplemented with 3% FBS |
mouse anti human CD3 antibody | eBioscience | 16-0037-81 | OKT3 clone |
mouse anti human CD28 antibody | ebioscience | 16-0288-81 | CD28.6 clone |
mouse anti human GD3 antibody | Abcam | ab11779 | R24 clone |
mouse anti human GD2 antibody | Santa Cruz Biotechnology | sc-53831 | 14G2a clone |
FITC-conjugated anti-mouse IgG3 antibody |
Abcam | ab97259 | |
Alexa Fluor 488 conjugated antimouse | Thermo Fisher Scieni¡tific | A-21131 | |
IgG2a antibody | |||
Alexa Fluor 647 conjugated antimouse | Thermo Fisher Scieni¡tific | A-21241 | |
IgG2a antibody | |||
Hoechst 333258 | Sigma-Aldrich | 861405 | |
Ficoll Paque-Plus | GE | 17-1440-02 | |
Trizol Reagent | Invitrogen | 15596-026 | |
Naive CD4+ T Cell Isolation Kit II, human | MACS Miltenyi Biotec | 130-094-131 | |
BD FACSAria II | BD Biosciences | Sorting | |
Nunc Lab-tek chamber slide system | Sigma-Aldrich | C7182 | |
Olympus FV 1000 Laser Confocal Microscope (Olympus) | Olympus | Upright BX61WI and IX81 | |
Forward sequence primer GD3 synthase | 5´-GAGCGTTCAGGAAACAAATGG- 3´ | Ref. 7 | |
Reverse sequence primer GD3 synthase | 5´-CCTGTGGGAAGAGAGAGTAAG-3´ | Ref. 7 | |
Forward sequence primer GM2/GD2 synthase | 5´-CAACACAGCAGACACAGTCC-3´ | Ref. 7 | |
Reverse sequence primer GM2/GD2 synthase | 5´-GTGGCAATCGTGACTAGAGC-3´ | Ref. 7 |