C1R cells expressing comparable levels of HLA-A2 D227K/T228A, wild-type HLA-A2, or HLA-A2 A245V/Kb were pulsed for 1 h with various concentrations of the indicated peptides. Cells were then washed twice with RPMI 1640 medium containing 100 U/mL penicillin and 100 μg/mL streptomycin and resuspended in R10. Each assay included 1.5 × 105 peptide-pulsed C1R cells and 5 × 104 MEL5 TCR+ CD8+ J.RT3-T3.5 cells. Unpulsed targets were used as negative controls. Expression of CD69 on the surface of MEL5 TCR+ CD8+ J.RT3-T3.5 cells was measured after 6 h using the following directly conjugated monoclonal antibodies: anti-CD8-α–PE-Cy7 (clone RPA-T8; Thermo Fisher Scientific), anti-CD8-β–eFluor660 (clone SIDI8BEE; Thermo Fisher Scientific), anti-CD69–BV421 (clone FN50; BioLegend), and anti-HLA-A2–FITC (clone BB7.2; BioLegend). Nonviable cells were excluded from the analysis using LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific). Data were acquired using a NovoCyte Flow Cytometer (ACEA Biosciences) and analyzed using FlowJo software version 10.6.1 (FlowJo LLC).
Anti cd8 β efluor660 clone sidi8bee
Manufactured by Thermo Fisher Scientific
The Anti-CD8-β–eFluor660 (clone SIDI8BEE) is a fluorescently-labeled monoclonal antibody that binds to the CD8-β subunit of the CD8 co-receptor. It is designed for use in flow cytometry applications to identify and characterize CD8-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions)
Novocyte flow cytometer, by Agilent Technologies (2 mentions)
Anti cd69 bv421 clone fn50, by BioLegend (2 mentions)
Anti cd8 α pe cy7 clone rpa t8, by Thermo Fisher Scientific (2 mentions)
Anti hla a2 fitc clone bb7.2, by BioLegend (2 mentions)
2 protocols using anti cd8 β efluor660 clone sidi8bee
1
HLA-A2 Variant Peptide Presentation
2
Peptide Pulsing and CD69 Expression Assay
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C1R cells expressing HLA-A2 D227K/T228A, wildtype HLA-A2, or HLA-A2 A245V/K b were pulsed for 1 h with various concentrations of the peptides ELTGIGILTV (3T), ELAGIGILTV (ELA), or FATGIGIITV (FAT). Cells were then washed twice with RPMI 1640 medium containing 100 U/ml penicillin and 100 mg/ml streptomycin and resuspended in R10. Each assay included 1.5 × 10 5 peptide-pulsed C1R cells and 5 x 10 4 MEL5 TCR + CD8 + J.RT3-T3.5 cells. Unpulsed targets were used as negative controls. Expression of CD69 on the surface of MEL5 TCR + CD8 + J.RT3-T3.5 cells was measured after 6 h using the following directly conjugated monoclonal antibodies: anti-CD8α-PE-Cy7 (clone RPA-T8; Thermo Fisher Scientific), anti-CD8β-eFluor660 (clone SIDI8BEE; Thermo Fisher Scientific), anti-CD69-BV421 (clone FN50; BioLegend), and anti-HLA-A2-FITC (clone BB7.2; BioLegend).
Non-viable cells were excluded from the analysis using LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific). Data were acquired using a NovoCyte Flow Cytometer (ACEA Biosciences) and analyzed using FlowJo software version 10.6.1 (FlowJo LLC).
Non-viable cells were excluded from the analysis using LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific). Data were acquired using a NovoCyte Flow Cytometer (ACEA Biosciences) and analyzed using FlowJo software version 10.6.1 (FlowJo LLC).
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