MV4;11 cells were counted on a Countess 3 Automated Cell Counter (Thermo Fisher, UK) with the addition of trypan blue. Cells (1x106) were aliquoted, spun down and resuspended in RPMI media containing test compounds at indicated concentrations. Additionally, QVD OPh (Sigma-Aldrich, UK) and Necrostatin-1 (Sigma-Aldrich) were added to the SIM1 treatment at 20μM final concentration. Treated cells were left to incubate for 24h at 37°C and 5% CO2. On the following day, the cells were collected in a Falcon tube and spun down at 500g for 5 min. Supernatant was aspirated and cells were washed once in 1mL FACS buffer (PBS, 5% FBS, 0.05 % NaN) and afterwards resuspended in 100μL of the same buffer containing Apotracker Green (Biolegend, UK) and DAPI (Sigma-Aldrich) at final concentration of 400nM and 1μg/mL, respectively. Cells were incubated for 20 min on ice and afterwards washed in 1mL of FACS buffer and finally resuspended in 500μL of the same buffer. Measurements were done on BD FACS Canto II flow cytometer (Flow Cytometry and Cell sorting facility, University of Dundee, UK) using blue (ex: 488 nm; em: 530±30 nm) and violet (ex: 405 nm; em: 450±50 nm) laser for detection of FITC and DAPI, respectively. Data were analysed on FlowJo™ 10.7.1. Software and GraphPad Prism. Gating strategy is detailed in Supplementary Figure 1.
10.7.1 software
Manufactured by FlowJo
FlowJo™ 10.7.1 is a software application designed for the analysis of flow cytometry data. It provides a comprehensive set of tools for data visualization, gating, and statistical analysis. The software is intended to assist researchers and scientists in the interpretation and management of flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Countess 3 automated cell counter, by Thermo Fisher Scientific (2 mentions)
Q vd oph, by Merck Group (2 mentions)
Apotracker green, by BioLegend (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Necrostatin 1, by Merck Group (2 mentions)
Hla dr bv421, by BD (1 mentions)
Cd69 pe, by Miltenyi Biotec (1 mentions)
Cd25 bv421, by BD (1 mentions)
Cd127 pe cf594, by BD (1 mentions)
Cd103 bv421, by BD (1 mentions)
Ccr7 pe cf594, by BD (1 mentions)
Navios cytometer, by Beckman Coulter (1 mentions)
3 protocols using 10.7.1 software
1
Flow Cytometry Analysis of Apoptosis
2
Comprehensive Phenotyping of Immune Cells
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The phenotype assay was performed as previously described [25] . In summary, we stained the cell surface for 20 min at 4°C using the following fluorochrome-conjugated antihuman antibodies: CD45RA FITC, CD27 APC, CD3 VioGreen, CD4 PECy7, CD8 APC Cy7 and L/D 7AAD. We employed other antibodies for specific cell populations: CD25 BV421 (BD Horizon, Franklin Lakes, NJ, USA) and CD127 PE-CF594 (BD Horizon) for regulatory T cells (Treg); HLA-DR BV421 (BD Pharmingen, San Diego, CA, USA), CD69 PE (Miltenyi Biotec) and CD25 BV421 (BD Horizon) for activation makers; CD279 (PD1) AF700 (BioLegend, San Diego, CA, USA) and NKG2A BV421 (BD OptiBuild) for exhaustion markers; and, CD103 BV421 (BD Horizon) and CCR7 PE-CF594 (BD Horizon) for chemokine receptor and integrin markers.
The experiments with and without IL-15 incubation O/N and with different concentrations of dexamethasone were performed as described previously. After the staining, cell acquisition was performed using a Navios cytometer (Beckman Coulter), acquiring a mean of 200,000 cells. The analysis was performed using FlowJo 10.7.1 software (FlowJo LLC).
The experiments with and without IL-15 incubation O/N and with different concentrations of dexamethasone were performed as described previously. After the staining, cell acquisition was performed using a Navios cytometer (Beckman Coulter), acquiring a mean of 200,000 cells. The analysis was performed using FlowJo 10.7.1 software (FlowJo LLC).
3
Flow Cytometry Analysis of Apoptosis
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MV4;11 cells were counted on a Countess 3 Automated Cell Counter (Thermo Fisher, UK) with the addition of trypan blue. Cells (1x106) were aliquoted, spun down and resuspended in RPMI media containing test compounds at indicated concentrations. Additionally, QVD OPh (Sigma-Aldrich, UK) and Necrostatin-1 (Sigma-Aldrich) were added to the SIM1 treatment at 20μM final concentration. Treated cells were left to incubate for 24h at 37°C and 5% CO2. On the following day, the cells were collected in a Falcon tube and spun down at 500g for 5 min. Supernatant was aspirated and cells were washed once in 1mL FACS buffer (PBS, 5% FBS, 0.05 % NaN) and afterwards resuspended in 100μL of the same buffer containing Apotracker Green (Biolegend, UK) and DAPI (Sigma-Aldrich) at final concentration of 400nM and 1μg/mL, respectively. Cells were incubated for 20 min on ice and afterwards washed in 1mL of FACS buffer and finally resuspended in 500μL of the same buffer. Measurements were done on BD FACS Canto II flow cytometer (Flow Cytometry and Cell sorting facility, University of Dundee, UK) using blue (ex: 488 nm; em: 530±30 nm) and violet (ex: 405 nm; em: 450±50 nm) laser for detection of FITC and DAPI, respectively. Data were analysed on FlowJo™ 10.7.1. Software and GraphPad Prism. Gating strategy is detailed in Supplementary Figure 1.
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