Fresh tumor samples were manually disaggregated between frosted-glass slides to obtain a single-cell suspension for analysis. Both the disaggregated tissue samples and expanded TIL were stained on ice in FACS Wash Buffer (Dulbecco’s Phosphate Buffered Saline 1× with 1% Bovine Serum Albumin) for 30 min with fluorochrome-conjugated monoclonal antibodies for CD3 FITC (SK7), CD4 PerCP-Cy5.5 (RPA-T4), CD8 PB (RPA-T8), CD16 PE (B159), CD28 PE-Cy7 (CD28.2), CD56 PE-Cy7 (B159), TCR y8 APC (B1), BTLA PE (J168 & J168–540), PD-1 BV650 (EH12) HLA-ABC PE (G46–2.6) (all BD Bioscience, San Jose, CA, USA), and PD-1 PerCP-Cy5.5 (EH12.2H7) (Biolegend, San Diego, CA, USA). Dead cells were excluded using the Aqua or Yellow LIVE/DEAD viability stain (ThermoFisher). Stained cells were fixed in 1% paraformaldehyde solution for 20 min at RT. Samples were acquired using the BD FACSCanto™ II or BD LSRFortessa X-20 and analyzed using FlowJo Software v10.5 (Tree Star). Subpopulations were excluded from analysis if less than 100 events.
Aqua or yellow live dead viability stain
Manufactured by Thermo Fisher Scientific
The Aqua or Yellow LIVE/DEAD viability stain is a fluorescent dye used to distinguish between live and dead cells. It provides a rapid and reliable method for assessing cell viability in a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BD (2 mentions)
Cd4 percp cy5, by BD (1 mentions)
Cd28 pe cy7, by BD (1 mentions)
Cd56 pe cy7, by BD (1 mentions)
Pd 1 percp cy5.5 eh12 2h7, by BioLegend (1 mentions)
Facs wash buffer, by BD (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd16 pe, by BD (1 mentions)
Pd 1 percp cy5, by BioLegend (1 mentions)
Cd8 pb rpa t8, by BD (1 mentions)
Cd28 pecy7 cd28, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
2 protocols using aqua or yellow live dead viability stain
1
Immune Profiling of Tumor Samples
2
Multiparametric Flow Cytometry for Expanded TILs
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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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