Immunophenotyping was undertaken following APC-conjugated HLA class I tetramer staining of PBMCs at 37°C for 15 min. Details of the tetramers used can be found in Supplementary Table 5 . Tetramers were conjugated to APC and a true tetramer response was verified through the lack of background staining by gating all CD3+ T cells, against CD8+ T cells and using a tetramer negative control. Surface staining with the following antibodies was then performed: live/dead blue dye (Invitrogen), anti-CD8 Amcyan (BD Biosciences), anti-CD3 APC-Cy7 (Biolegend), anti-PD-1 PercpCy5.5 (BD Biosciences), anti-CTLA4 PE-Cy7, anti-CD244 FITC, and anti-CD160 PE (Biolegend) before washing and flow cytometric analysis. Memory subset analysis utilized the same panel but included anti-CCR7 FITC (R&D systems) and anti-CD45RA AF700 (Biolegend) and omitted anti-CTLA4, anti-CD244, and anti-CD160. Example flow plots can be found in (Figure S1 ).
Anti cd244 fitc
Manufactured by BioLegend
Anti-CD244 FITC is a fluorescently labeled antibody that binds to the CD244 cell surface protein. CD244, also known as 2B4, is a member of the signaling lymphocyte activation molecule (SLAM) family of receptors and is expressed on natural killer cells, CD8+ T cells, and other immune cell types. The FITC (fluorescein isothiocyanate) fluorescent label allows for the detection and analysis of CD244-expressing cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Anti cd8 amcyan, by BD (1 mentions)
Anti ccr7 fitc, by R&D Systems (1 mentions)
Anti cd3 apc cy7, by BioLegend (1 mentions)
Anti cd160 pe, by BioLegend (1 mentions)
Live dead blue dye, by Thermo Fisher Scientific (1 mentions)
Anti pd 1 percpcy5, by BD (1 mentions)
Anti ctla4 pe cy7, by BioLegend (1 mentions)
Anti cd45ra af700, by BioLegend (1 mentions)
Monensin, by Merck Group (1 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Anti cd57 vioblue, by Miltenyi Biotec (1 mentions)
Anti il 10 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd8 pe vio770, by Miltenyi Biotec (1 mentions)
Anti cx3cr1 pe, by Thermo Fisher Scientific (1 mentions)
Cyan adp, by Beckman Coulter (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
2 protocols using anti cd244 fitc
1
Detailed Immunophenotyping of T Cell Subsets
2
Multiparameter Flow Cytometry Assay
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All cells were assessed for viability using a fixable viability dye (ebioscience eFluor506). Flow cytometric analysis of human and murine cells was performed using >50 antibodies and appropriate directly conjugated mouse, rat and hamster isotype controls. The clones . relevant to the majority of work are as follows: mouse anti-CD3-FITC (Miltenyi; REA641) anti-CD8a-PerCP (Miltenyi; 53-6.7) anti-CD28-PE (Miltenyi; 37.51) anti-KLRG1-APC (Miltenyi; 2F1). Human anti-CD3 APC-Cy7 (biolegend; HIT3a) anti-CD8-PE-Vio770 (Miltenyi; REA734) anti-CD28-eFluor450 (ebioscience; CD28.2) anti-KLRG1-AF488 (a generous gift from Professor Hanspeter Pircher, University of Freiburg) anti-CD57-Vioblue (Miltenyi; TB03) anti-CD244-FITC (biolegend; C1.7) anti-CX3CR1-PE (ebioscience; 2A9-1), and anti-IL-10-FITC (ebioscience; BT-10). For intracellular cytokine staining, cells were cultured with PMA (50 ng/ml), ionomycin (500 ng/ml), and monensin (3 µM; all from Sigma) for 4 h at 37C. Cells were stained for cell surface markers, then fixed and permeabilized in Cytofix/Cytoperm (BD) before intracellular detection of cytokines. Cells were acquired using a CyAn ADP (Beckman Coulter) flow cytometer and analysed using Summit software (software version 4.3; Beckman Coulter). Percentages and mean fluorescence intensity (MFI) were calculated against appropriate isotype controls.
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