Cells were blocked in anti-CD16/CD32 (eBioscience) prior to staining with anti-CD11b-V450, CD19-PE or Gr1-PE, CD3-PE-Cy™7, CD45-PerCP-Cy™5.5 or CD19-PerCP-Cy™5.5, Ly-6C-APC (all from BD) and F4/80-FITC (eBioscience, Hatfield, UK). Lamina propria cells were incubated with the LIVE/DEAD® stain (Invitrogen) prior to staining above. Cells were run on a BD LSRFortessa or LSR II after optimisation with compensation particles (BD) and analysed using FlowJo (Tree Star, Ashland, OR, USA).
Compensation particles
Manufactured by BD
Sourced in United States
Compensation particles are a type of laboratory equipment used in flow cytometry analysis. Their primary function is to help calibrate and adjust the sensitivity of flow cytometers, ensuring accurate and reliable measurements of cellular characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b v450, by BD (1 mentions)
Cd19 percp cy5, by BD (1 mentions)
Ly6c apc, by BD (1 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
Anti cd16 cd32, by Thermo Fisher Scientific (1 mentions)
F4 80 fitc, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cd3 pe cy7, by BD (1 mentions)
Cd45 percp cy5, by BD (1 mentions)
Cd3 percp, by BD (1 mentions)
Ifn γ apc, by Thermo Fisher Scientific (1 mentions)
Tnfα fitc, by Thermo Fisher Scientific (1 mentions)
Live dead fixable aqua dye, by Thermo Fisher Scientific (1 mentions)
Cd8 pb, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Cd4 pe cy7, by BD (1 mentions)
Zombie red fixable viability kit, by BioLegend (1 mentions)
Brain tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Fluorochrome conjugated anti mouse antibodies, by BioLegend (1 mentions)
4 protocols using compensation particles
1
Multicolour Flow Cytometry Analysis
2
T-cell Response Profiling Protocol
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Briefly, cells were incubated for 6 h at 37°C in 5% CO2 with pools of overlapping 15-mer long peptides at a final concentration of 2 μM each, stimulated with SEB (Sigma) at 1 μg/ml or left unstimulated (negative control). The secretion inhibitor brefeldin A (Sigma) was added for the final 5 h of culture. After the 6 h incubation, surface staining for CD3-PercP, CD4-PECy7, CD8-PB (unless otherwise stated, staining reagents, instrumentation and software were from BD Biosciences) and live/dead fixable aqua dye (Life Technologies) was performed and then followed by intracellular staining for IFNγ-APC, TNFα-FITC, MIP-1β-PE, CD154-APC-eFluor780 (eBiosciences, San Diego, CA). Analysis was done on a two-laser CANTO II flow cytometer (BD Biosciences) and data analyzed with FlowJo software (TreeStar Inc., Ashland, OR). Spectral compensation was performed for each experiment with each individual mAb used in the surface and intracellular cytokines staining using compensation particles (BD Biosciences).
3
Multicolor Flow Cytometry of Brain Tumors
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For multicolor flow cytometric analysis, brain tumor quadrants were harvested, minced, incubated with a Brain Tumor Dissociation Kit (130-095-942, Miltenyi), triturated, passed through a 70 mm screen, resuspended in FACS buffer (2% inactivated fetal calf serum in PBS), and stained with fluorochrome-conjugated anti-mouse antibodies from BioLegend. A Zombie Red Fixable viability kit (BioLegend) was used to stain dead cells. We followed a ‘no-wash’ sequential staining protocol (BioLegend) to stain dead cells and for surface staining. Intracellular Foxp3 staining was performed following the Foxp3 intracellular staining protocol (BioLegend). For single-color compensation controls, compensation particles (552845, BD) were used and stained with each fluorescently conjugated antibodies according to the manufacturer’s instructions. For the Zombie Red assay, cells from the nontumor and tumor quadrants, respectively, were used as single-color compensation controls. All samples were run in a Cytoflex flow cytometer (Beckman Coulter). Data were analyzed with CytExpert software. Technicians acquiring and gating the data were blinded to the treatments.
4
Multicolor Flow Cytometry-based Lysis Assay
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CD4-PacificBlue (clone OKT4; 1:100), CD8-FITC (clone RPA-T8; 1:100), CD10-APC/Cy7 (clone HI10a, 1:100), CD69-PE (clone FN50; 1:200), CD276-PE/Cy7 (clone MIH42; 1:100) and the respective isotype control antibodies were purchased from BioLegend (San Diego, USA). CD45-AmCyan (clone 2D1, 1:200) was purchased from BD Biosciences (Franklin Lakes, USA).
For flow cytometry-based lysis assays, 100,000 target cells were incubated in 96 well plates together with 100,000 PBMCs, blinatumomab at 1 ng/ml and different tyrosine kinase inhibitors at the indicated concentrations. After 3 days, flow cytometric analysis was performed. Nalm-6 cells were defined as CD45−CD10+, Nalm-16 as CD45−CD10+, SD-1 per cell size and as CD22+, TOM-1 as CD45−CD10+, T cells as CD45+CD4+ or CD45+CD8+, and activated subsets were identified using CD69 as surrogate marker. Absolute cell numbers were determined by the acquisition of equal amounts of compensation particles (BD Biosciences) per sample, thus allowing for calculation of the absolute degree of tumor cell lysis. Binding of antibodies to Fc receptors was blocked with Flebogamma DIF (Grifols, Barcelona, Spain) at 50 μg/ml. Data acquisition was performed using a FACSCanto II (BD Biosciences). For data analysis, FlowJo_V10 software (Tree Star, Ashland, OR) was utilized.
For flow cytometry-based lysis assays, 100,000 target cells were incubated in 96 well plates together with 100,000 PBMCs, blinatumomab at 1 ng/ml and different tyrosine kinase inhibitors at the indicated concentrations. After 3 days, flow cytometric analysis was performed. Nalm-6 cells were defined as CD45−CD10+, Nalm-16 as CD45−CD10+, SD-1 per cell size and as CD22+, TOM-1 as CD45−CD10+, T cells as CD45+CD4+ or CD45+CD8+, and activated subsets were identified using CD69 as surrogate marker. Absolute cell numbers were determined by the acquisition of equal amounts of compensation particles (BD Biosciences) per sample, thus allowing for calculation of the absolute degree of tumor cell lysis. Binding of antibodies to Fc receptors was blocked with Flebogamma DIF (Grifols, Barcelona, Spain) at 50 μg/ml. Data acquisition was performed using a FACSCanto II (BD Biosciences). For data analysis, FlowJo_V10 software (Tree Star, Ashland, OR) was utilized.
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