In brief, healthy donors and patient’s total PBMC were stained for surface markers with the following fluorochrome conjugated antibodies: CD19-BUV737, CD107a-BUV395, CD3-BV786, CD33-BV711 & CD14-BV650 (for AML study), CD15-BV650 & CD30-BV605 (for HL study), PD1-FITC, CD62L-PerCP-Cy5.5, CD57-PE-CF594, CD16-AF700 from Beckton Dickinson, CD69-PE from Beckman Coulter, CD45RO-VioGreen, CD7-VioBlue, CD56-PE-Vio770, CD45RA-APC-Vio770, and NKG2C-APC from Miltenyi Biotec. Cell viability was determined using DAPI (BD Biosciences). Cells were stained with the corresponding antibodies in FACS buffer (PBS, 2% FBS) on ice for 25–30 min and washed twice with the same FACS buffer and acquired on BD LSR-Fortessa instrument (Blue-Yellow/Green-Red-Violet-Ultraviolet) (BD Bioscience). Flow Cytometry Standard (FCS) files were analyzed using FlowJo software v10.6.1 (Becton, Dickinson and Company, Ashland, OR). The gating strategy for conventional flow cytometry and the determination of the “percentage” of selected cells in the figures is described in the Supplemental Figure S1 .
Cd45ra apc vio770
Manufactured by Miltenyi Biotec
The CD45RA-APC-Vio770 is a fluorescently labeled antibody that binds to the CD45RA antigen. CD45RA is a cell surface marker expressed on naïve T cells and a subset of memory T cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd45ro viogreen, by Miltenyi Biotec (3 mentions)
Nkg2c apc, by Miltenyi Biotec (2 mentions)
Lsr fortessa instrument blue yellow green red violet ultraviolet, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (2 mentions)
Nkp46 pe vio770, by Miltenyi Biotec (2 mentions)
Pd1 fitc, by BD (1 mentions)
Cd69 pe, by Beckman Coulter (1 mentions)
Cd56 pe vio770, by Miltenyi Biotec (1 mentions)
Cell staining buffer, by BioLegend (1 mentions)
Ccr4 pe, by BioLegend (1 mentions)
Fitc pe, by BD (1 mentions)
Cd28 percp cy5.5, by Thermo Fisher Scientific (1 mentions)
Pd 1 fitc clone eh12.2h7, by BioLegend (1 mentions)
Tim 3 apc cy7, by BioLegend (1 mentions)
Cxcr2 pe cy7, by BioLegend (1 mentions)
Cxcr3 fitc, by BioLegend (1 mentions)
Goat anti mouse f ab 2 antibody anti fab, by Jackson ImmunoResearch (1 mentions)
Ccr2 apc, by BioLegend (1 mentions)
Vioblue, by Miltenyi Biotec (1 mentions)
Cd8 pe cy7, by BioLegend (1 mentions)
4 protocols using cd45ra apc vio770
1
Multiparametric Flow Cytometry Profiling of PBMCs
2
Characterization of CAR-T Cells by Flow Cytometry
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The following antibodies were used: CD3 (VioBlue; Miltenyi Biotech or Pacific blue and PE; BioLegend), CD4 (FITC or APC-Cy7; BioLegend), CD8 (PE-Cy7; BioLegend), CD3/CD19 antibodies (FITC/PE; BD), CD28 (PerCP-Cy5.5; eBioscience), PD-1 (FITC; clone: EH12.2H7; BioLegend), TIM-3 (APC-Cy7; BioLegend), LAG-3 (VioBlue; Miltenyi Biotech), CD45RA (APC-Vio770; Miltenyi Biotec or Brilliant Violet; BioLegend), CCR7 (PerCP-Vio770; Miltenyi Biotec or PerCP; BioLegend), CCR2 (APC; Biolegend), CCR4 (PE; Biolegend), CCR5 (Alexa Influenza 488; Biolegend), CXCR2 (PE-Cy7; Biolegend) and CXCR3 (FITC; Biolegend).
Transduction efficacy was determined on day 6 and day 9 of culture by labeling CAR-T cells with biotin-labeled polyclonal goat anti-mouse F(ab)2 antibody (anti-Fab, Jackson Immunoresearch, West Grove, Pennsylvania) and streptavidin (APC conjugated; BioLegend). CD3+F(ab)2+ cells were defined as CAR-T cells. Isotype labeled cells (Jackson Immunoresearch) and untransduced cells served as negative controls. For further characterization, cells were stained with antibodies mentioned above. Cells were washed and re-suspended in cell staining buffer (BioLegend), incubated for 30 min with the antibodies on ice, washed and measured using MACSQuant FACS cytometer (Miltenyi Biotec). Samples were analyzed by FlowJo software (FlowJo LLC, Ashland, Oregon).
Transduction efficacy was determined on day 6 and day 9 of culture by labeling CAR-T cells with biotin-labeled polyclonal goat anti-mouse F(ab)2 antibody (anti-Fab, Jackson Immunoresearch, West Grove, Pennsylvania) and streptavidin (APC conjugated; BioLegend). CD3+F(ab)2+ cells were defined as CAR-T cells. Isotype labeled cells (Jackson Immunoresearch) and untransduced cells served as negative controls. For further characterization, cells were stained with antibodies mentioned above. Cells were washed and re-suspended in cell staining buffer (BioLegend), incubated for 30 min with the antibodies on ice, washed and measured using MACSQuant FACS cytometer (Miltenyi Biotec). Samples were analyzed by FlowJo software (FlowJo LLC, Ashland, Oregon).
3
Comprehensive Immunophenotyping of Blood Cells
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In brief, healthy donors and patient’s total peripheral blood mononuclear cells (PBMC) were stained for surface markers with the following fluorochrome conjugated antibodies: CD4-BUV737, CD56-BUV395, CD3-BV786, CD16-BV711, CD7-BV421, HER2-BV650, CD326-BV605, PD1-FITC, MUC1-PerCP, CD107a-PE-CF594, CD14-AF700, CD19-AF700, PSMA-PE, CD45RO-VioGreen, NKp46-PE-Vio770, CD45RA-APC-Vio770 and CD138-APC (all from Miltenyi Biotec). Cell viability was determined using DAPI exclusion (BD Biosciences). Cells were stained with antibodies cocktail in FACS buffer at 4°C for 25-30 min and washed twice with the same FACS buffer and acquired on BD LSR-Fortessa instrument (Blue-Yellow/Green-Red-Violet-Ultraviolet) (BD Bioscience). FCS files were analyzed using FlowJo software v10.6.1 (Tree Star Inc.).
4
Multiparametric Flow Cytometry for Immune Monitoring
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For immune-monitoring studies, patient PBMC samples were stained with cocktail fluorescent-conjugated antibody mix diluted in PBS containing 2% FBS + 0.01% azide for 30 min at 4 °C in the dark. After a 2-step washing with PBS, cells were fixed with 2% PFA for 20 min at 4 °C. Samples were analyzed on a BD Fortessa Flow Cytometer (UV-Violet-Blue-Yellow/Green-Red 5-Laser configuration). For the first cohort (aviremic patients) the following antibodies were used: CCR5-FITC, CD158a-PE, CD57-PE-CF594, CD14-Alexa Fluor 700, CD19-Alexa Fluor 700, CXCR4-BV421, CD62L-BV605, NKG2D-BV650, CD16-BV711, CD3-BV786, CD56-BUV395 and CD4-BUV737 (BD Biosciences), CD158b-PE, CD158e/k-PE, NKp46-PE-Vio770, NKG2C-APC, CD45RA-APC-Vio770, and CD45RO-VioGreen (Miltenyi). For the second cohort (viremic patients) the similar panel of antibodies were re-used, except for additional antibodies for CXCR4-BV421 and CD107a-PE-CF594 (BD Biosciences). Cell death and viability were determined by DAPI (BD Biosciences). Data were analyzed using FlowJo software (v10.8) (BD Biosciences) with appropriate plugins for high-dimensional analysis and visualization.
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