Spleens were embedded in Optimal Cutting Temperature (O.C.T.) compound (Fisher Scientific) and snap frozen in the vapor phase of liquid nitrogen. 15 μm sections were generated then fixed with 100% ice cold acetone followed by blocking with PBS/5%BSA. Sections were stained overnight with anti-CD8α-FITC (53-6.7; eBioscience), anti-IgD-PE-Dazzle594 (11-26c.2a, Biolegend), and anti-GL7-eFluor450 (GL-7; eBioscience) and single plane confocal imaged on a Zeiss LSM 880 confocal system with a 10× objective. Confocal images were processed in ImageJ to adjust for contrast and pseudocolored in red, green, and blue. GL-7+ GCs were traced in ImageJ using the freehand selection tool and the area was determined. CD8+ cells within the GC were marked using the multi-point selection tool when a dark center (nucleus) surrounded by CD8 surface staining could be identified. GC CD8 T cells within GC area were quantified manually.
Anti cd8α fitc 53 6.7
Manufactured by Thermo Fisher Scientific
Anti-CD8α-FITC (53-6.7) is a fluorescently-labeled antibody that binds to the CD8α chain, a surface marker expressed on a subset of T cells. It can be used to identify and quantify CD8-positive cells in flow cytometry applications.
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Lab products found in correlation
Oct compound, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal system, by Zeiss (1 mentions)
Anti cd19 pe vio770, by Miltenyi Biotec (1 mentions)
Macs cytokine secretion assay, by Miltenyi Biotec (1 mentions)
Anti cd3 percp efluor 710, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facscanto, by BD (1 mentions)
2 protocols using anti cd8α fitc 53 6.7
1
Immunofluorescence Analysis of Germinal Center CD8 T Cells
2
Multiparameter Flow Cytometry of Immune Cells
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All antibodies were titrated for optimal staining prior to experimentation. The following antibodies were used for surface staining (obtained from Biolegend unless otherwise indicated): anti-CD3-PerCP-eFluor710 (17A2; eBioscience), anti-CD19-PE-Vio770 (6D5; Miltenyi Biotec), anti-CD4-Pacific Blue (GK1.5), anti-CD8α-VioGreen (53–6.7; Miltenyi Biotec), anti-CD8α-FITC (53–6.7), anti-PD-1-PE (RMP1-30), anti-Ki-67-FITC (SolA15; eBioscience) and anti-CD62L-biotin (MEL-14; BD Pharmingen). Viability was determined in some samples using Fixable Viability Dye eFluor780 (eBioscience). For identification of IFN-γ or IL-10 secreting cells by flowcytometry, the MACS Cytokine Secretion Assay (Miltenyi Biotec) was used. Streptavidin conjugated antibodies were detected using biotinylated PE or DyeLight 488 (Biolegend). The samples were analyzed on a BD FACSCanto or LSR Fortessa flow cytometer (BD biosciences, San Jose, CA, USA) (by gating on viable cells and using FCS express software version 3.3 (DeNovo Software, Los Angeles, CA, USA). Approximately between 104 and 2 × 105 cells were analyzed from each sample.
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