Cryopreserved specimens were thawed, washed, and counts and viability were assessed using the Countless Automated Cell Counter system (Invitrogen, Carlsbad, CA). PBMC were washed and stained in Brilliant Violet Stain Buffer (BD Biosciences, San Jose, CA) at room temperature for 15 min in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry data acquisition. mAbs used in flow cytometry; CD3 AF700 (clone UCHT1), CD4 PE-CF594 (clone RPA-T4), CD8 BV711 (clone RPA-T8), CD38 APC-H7 (clone HB7), CD161 BV421 (clone DX12), HLA-DR APC (clone L243), TCR γδ BV650 (clone B1), and PD-1 PE-Cy7 (clone EH12.1) were all from BD Biosciences, TCR Vδ1 FITC (clone TS8.2) was from Abcam (Cambridge, MA), and TCR Vδ2 PE (clone B6) was from Biolegend (San Diego, CA). Live/dead aqua fixable cell stain was from Life Technologies (Eugene, OR). All antibodies were used together in 1 panel. A minimum number of 200 events were recorded for all subsets of γδ T cells. Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar, Ashland, OR).
Countless automated cell counter system
Manufactured by Thermo Fisher Scientific
The Countless Automated Cell Counter system is a lab equipment product that provides automated cell counting capabilities. It is designed to accurately determine the number of cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Lsr fortessa instrument, by BD (2 mentions)
Brilliant violet stain buffer, by BD (2 mentions)
Pd 1 pe cy7 clone eh12.1, by BD (2 mentions)
Live dead aqua fixable cell stain, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Cd8 bv711, by BD (1 mentions)
Cd4 bv605, by BD (1 mentions)
Tcr vα7.2 percp cy5 5 clone 3c10, by BioLegend (1 mentions)
Plzf apc, by R&D Systems (1 mentions)
Cd3 percp cy5, by BD (1 mentions)
Perm wash, by BD (1 mentions)
Cd3 af700, by BD (1 mentions)
2 protocols using countless automated cell counter system
1
Multiparametric Immunophenotyping of Cryopreserved PBMCs
2
Comprehensive Immunophenotyping of Cryopreserved PBMCs
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Cryopreserved PBMC specimens were thawed and washed, and counts and viability were assessed using the Countless Automated Cell Counter system (Invitrogen, Carlsbad, CA). Cells were washed and stained in Brilliant Violet Stain Buffer (BD Biosciences, San Jose, CA) at room temperature for 15 min in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry data acquisition. Intracellular staining for IFNγ and promyelocytic leukaemia zinc finger (PLZF) were performed in Perm/Wash (BD Biosciences) at room temperature for 15 min in the dark. mAbs used in flow cytometry: CD3 AF700, CD3 PerCP-Cy5.5 (both clone UCHT1), CD4 BV605 (clone RPA-T4), CD8 BV711 (clone RPA-T8), CD38 APC-H7 (clone HB7), CD69 AF00 (clone FN50de), CD161 BV421 (clone DX12), CCR6 BV786 (clone 11A9), HLA-DR APC (clone L243), IFNγ APC (clone B27), and PD-1 PE-Cy7 (clone EH12.1) were all from BD Biosciences, TCR Vα7.2 PE, TCR Vα7.2 PerCP-Cy5.5 (clone 3C10) were from Biolegend (San Diego, CA, USA), and PLZF APC was from R&D Systems (Minneapolis, MN). Live/dead aqua fixable cell stain was from Life Technologies (Eugene, OR, USA) and added to the cells together with the cell surface antibodies. Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar, Ashland, OR, USA).
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