Blood or brain single-cell suspensions were washed in PBS supplemented with 1% FBS, 2 mM EDTA and 0.01% sodium azide, and incubated for 10 min at 4 • C with culture supernatant from the hybridoma cell line 2.4G2 to block Fc receptors. The cells were surface stained with fluorochrome-conjugated monoclonal antibody for 15 min at 4 • C. LIVE/DEAD fixable blue dye (Life Technologies) was used to exclude dead cells. The following antibodies were used: anti-CD45.1 (clone A20, BioLegend), anti-CD11b (clone M1/70, BD), anti-CD19 (clone 6D5, BioLegend), anti-CD3 (clone 145-2C11, BioLegend), anti-CD4 (clone RM4-5, BD), anti-CD8 (clone 53-6.7, BD or Biolegend), anti-FOXP3 (clone FJK-16s, eBioscience), anti-CD44 (clone IM7, BioLegend), anti-CD62L (clone MEL-14, BD), anti-CD127 (clone A7R34, BioLegend) and anti-KLRG1 (clone 2F1, eBioscience). OVA-specific CD8 + T cells were detected using H-2K b /SIINFEKL pentamers (ProImmune). Intracellular FoxP3 staining was performed using BioLegend FOXP3 Fix/Perm Buffer Set. Samples were acquired on LSRII Flow Cytometer (BD), and the data were analyzed using FlowJo software (TreeStar Inc.).
H 2k b siinfekl pentamers
Manufactured by ProImmune
Sourced in United Kingdom
H-2K b /SIINFEKL pentamers are a type of lab equipment used for the detection and analysis of specific immune cells. They are composed of MHC class I molecules loaded with the SIINFEKL peptide, which can be used to identify and study T cells that recognize this peptide-MHC complex.
Automatically generated - may contain errors
Lab products found in correlation
Anti foxp3 clone fjk 16s, by Thermo Fisher Scientific (1 mentions)
Anti cd8 clone 53 6, by BD (1 mentions)
Anti cd3 clone 145 2c11, by BioLegend (1 mentions)
Anti cd11b clone m1 70, by BD (1 mentions)
Anti cd45.1 clone a20, by BioLegend (1 mentions)
Anti cd127 clone a7r34, by BioLegend (1 mentions)
Anti cd62l clone mel 14, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Live dead fixable blue dye, by Thermo Fisher Scientific (1 mentions)
Anti cd19 clone 6d5, by BioLegend (1 mentions)
Foxp3 fix perm buffer set, by BioLegend (1 mentions)
Anti cd44 clone im7, by BioLegend (1 mentions)
Anti klrg 1 clone 2f1, by Thermo Fisher Scientific (1 mentions)
Anti cd4 clone rm4 5, by BD (1 mentions)
Cyan adp, by Beckman Coulter (1 mentions)
Anti tnf antibody, by BD (1 mentions)
Ethidium monoazide bromide, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti ifnγ xmg1.2, by Thermo Fisher Scientific (1 mentions)
2 protocols using h 2k b siinfekl pentamers
1
Multiparametric Flow Cytometry Analysis
2
Multicolor Flow Cytometry for T-cell Analysis
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Anti-CD4 (GK1.5), anti-CD8β (H35–17.2), anti-CD44 (IM7), anti-CD62L (MEL-14) and anti-IFNγ (XMG1.2) antibodies were purchased from eBioscience or BD PharMingen. OVA-specific CD8+ T cells were visualized by staining cells with H-2Kb/SIINFEKL pentamers (ProImmune, Oxford, UK). Anti-TNF antibody was purchased from BD PharMingen. Intracellular cytokine staining was performed after fixation with 2% paraformaldehyde and permeabilization with 0.5% Saponin.
Cells were acquired on LSRII (BD, Biosciences) or CyAn™ ADP (Beckman Coulter). Data analysis was performed with FlowJo software (Tree Star). Dead cells were excluded by PI, DAPI or ethidium bromide monoazide (Sigma).
Cells were acquired on LSRII (BD, Biosciences) or CyAn™ ADP (Beckman Coulter). Data analysis was performed with FlowJo software (Tree Star). Dead cells were excluded by PI, DAPI or ethidium bromide monoazide (Sigma).
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