The freshly collected BAL cells were stimulated ex vivo with Mtb-specific antigens, ESAT-6/CFP-10 and Mtb Cell Wall Fraction (BEI Resources, 10 μg/mL) for a total of 16 h. Brefeldin A (0.5 μg/mL, SIGMA) was added 2 h after the onset of stimulation. After stimulation, the cells were stained with LIVE/DEAD fixable Near-IR stain (ThermoFisher) and stained subsequently with the surface antibodies: CD4-PerCP-Cy5.5 (L200, BD Biosciences), CD8-APC (RPA, T8, BD Biosciences), CD3-AlexaFlour 700 (SP34 2, BD Biosciences), CD95-BV421 (DX2, BD Biosciences), CD28-PECy7 (CD28.2, BD Biosciences) and CD45-BUV395 (D058 1283, BD Biosciences). Cells were then fixed, permeabilized and stained with intracellular antibodies: IFNγ-APC-Cy7 (B27, Biolegend), IL-17-BV605 (BL168, Biolegend) and TNF-α-BV650 (MAb11, Biolegend). Cells were washed, suspended in BD stabilizing fixative buffer and acquired on BD Symphony flow cytometer. Analysis was performed using FlowJo (v10.6.1) using previously published gating strategy (18 (link)–20 (link)) (Figures S1 – S3
Cd28 pecy7 cd28
Manufactured by BD
Sourced in United States
CD28-PECy7 (CD28.2) is a fluorochrome-conjugated antibody that binds to the CD28 receptor expressed on the surface of T cells. CD28 is a co-stimulatory molecule that plays a critical role in T cell activation and proliferation. The PECy7 fluorochrome allows for the detection and analysis of CD28-positive cells using flow cytometry.
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Lab products found in correlation
Live dead fixable near ir stain, by Thermo Fisher Scientific (1 mentions)
Cd4 percp cy5.5 l200, by BD (1 mentions)
Cd8 apc rpa t8, by BD (1 mentions)
Symphony flow cytometer, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Pd 1 percp cy5, by BioLegend (1 mentions)
Aqua or yellow live dead viability stain, by Thermo Fisher Scientific (1 mentions)
Cd8 pb rpa t8, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Cd3 fitc, by BD (1 mentions)
2 protocols using cd28 pecy7 cd28
1
Mtb-specific T cell Responses Ex vivo
2
Multiparametric Flow Cytometry for Expanded TILs
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Expanded TIL were first washed in FACS Wash Buffer (Dulbecco’s phosphate-buffered saline 1X (PBS, Thermo Fisher Scientific)) with 1% bovine serum albumin (Millipore Sigma). Surface Fc receptors were blocked for 10 min at room temperature using goat serum (Sigma) diluted in FACS Wash Buffer (5%) before proceeding with surface staining on ice (100 µL per reaction) for 30 min. Cell surface expression assessment for this study was done using fluorochrome-conjugated antibodies against CD3 FITC (SK7), CD4 PerCP-Cy5.5 (RPA-T4), CD4 BUV496 (SK3), CD28 PE-Cy7 (CD28.2), CD8 PB (RPA-T8) (all BD Bioscience, San Jose, California, USA), LAG3 PE (3DS223H) (Life Technologies, Carlsbad, California, USA), PD-1 PerCP-Cy5.5 (EH12.2H7), CD27 APC (M-T271), CD8 APC-Cy7 (SK1) (Biolegend, San Diego, California, USA). Aqua or Yellow Live/Dead viability stain (Thermo Fisher Scientific) was used to exclude dead cells from analysis. Stained cells were fixed with 1% paraformaldehyde (Electron Microscope Sciences, Hatfield, Pennsylvania, USA) solution for 20 min at room temperature.
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