At experimental endpoint of the intracranial tumor experiments in which mice were treated with CAR T cells, some organs were taken for analysis by flow cytometry. The brain, spleen, lymph nodes and blood (in the form of a cardiac bleed) were isolated from mice from each treatment group (n = 3 or 4 mice per group). Organs were processed through a 70‐µm filter to a single‐cell suspension and prepared for antibody staining. Blood was collected in a heparin/EDTA tube. Red blood cells were lysed by incubation in red cell removal buffer (156 mm ammonium chloride, 11.9 mm sodium bicarbonate, 0.097 mm EDTA, WEHI, Melbourne) and centrifugation at 548RCF for 5 min at 4°C. Cells were labelled with a cell surface murine antibody cocktail containing anti‐CD3‐AF700 (BD Pharmingen, New Jersey), anti‐CD4‐BV421 (BD Horizon, New Jersey), anti‐CD8‐BV711 (BioLegend, San Diego), anti‐PD‐1 (BD Pharmingen, New Jersey), LAG‐3 (CD223)‐APC (BD Pharmingen, New Jersey), anti‐CD27‐BV605 (BioLegend, San Diego), anti‐CD28‐PE‐Cy7 (eBioscience, San Diego), CD44‐BV786 (BD Horizon, New Jersey) and anti‐CD62L‐BV510 (BD Horizon, New Jersey). Samples were analysed using a BD Fortessa X20 flow cytometer and FlowJo™ software v10 (BD, New Jersey).
Anti cd8 bv711
Manufactured by BioLegend
Sourced in United States
Anti-CD8-BV711 is a fluorochrome-conjugated antibody that binds to the CD8 receptor expressed on the surface of certain T cells. It is designed for use in flow cytometry applications to identify and quantify CD8-positive cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 af700, by BD (2 mentions)
Fortessa x20 flow cytometer, by BD (2 mentions)
Anti cd4 bv421, by BD (2 mentions)
Anti cd27 bv605, by BioLegend (1 mentions)
Anti pd 1, by BD (1 mentions)
Anti cd3 bv711, by BioLegend (1 mentions)
Anti cd45ra bv421, by BioLegend (1 mentions)
Anti cd4 bv785, by BioLegend (1 mentions)
Anti cd45ro pecy7, by BioLegend (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Anti cd19 pe, by BioLegend (1 mentions)
Anti cd27 pe, by BioLegend (1 mentions)
Anti cd45 bv711, by BioLegend (1 mentions)
Anti ccr7 apc, by BioLegend (1 mentions)
Streptavidin pe, by BioLegend (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Clone mp6 xt22, by BioLegend (1 mentions)
Anti ifn γ apc, by BD (1 mentions)
Anti il 2 pe, by BD (1 mentions)
5 protocols using anti cd8 bv711
1
Phenotypic Analysis of CAR T Cell Therapy
2
Comprehensive CAR Expression Analysis
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For evaluation of CAR expression, cells were stained with a goat anti-mouse Fab antibody conjugated with Alexa Fluor 647 (Jackson ImmunoResearch) or biotinylated CD19-Fc (ACRO Biosystems) for 15 min at room temperature. Cells were thoroughly washed before staining with antibodies for additional surface markers. Anti-CD3-BB515 and anti-CD62L-BB515 were purchased from BD Biosciences. Anti-CD4-BV785, anti-CD8-BV711, anti-CD45RA-BV421, anti-CD45RO-PECy7, streptavidin-PE, anti-CD27-PE, anti-CD95-allophycocyanin (APC)Cy7, anti-CCR7-APC, anti-CD45-BV711, and anti-CD19-PE, anti-CD3-BV711 were purchased from BioLegend and used to stain surface antigens. Cell Trace Violet was purchased from Thermo Fisher Scientific and used at a concentration of 1 μM to label cells for 10 min. All data were acquired using a BD Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software.
3
Cytokine profiling of CAR T cells
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CAR T cells and tumour cells were co-incubated for 5 h in T cell media at a 1:1 ratio in the presence of Golgi-stop (Becton Dickinson, Kit #554715, final concentration 5 ml/mL), at 37 °C, 5% CO2. Cells were subsequently incubated with antibodies to surface proteins in FACS buffer (Phosphate Buffered Saline (PBS) with 0.2% Bovine Serum Albumin (BSA, Gibco)), with anti-CD4-BV421 (Clone MP6-XT22, BD Horizon, Franklin Lakes, NJ, USA), anti-CD8-BV711 (Clone 53-6.7, BioLegend, San Diego, CA, USA), and the viability dye Fixable yellow (Invitrogen, Oregon) for 30 min at 4 °C. Cells were then fixed for 20 min at room temperature and permeabilised using BD Pharmingen Fix/Perm kit (Cat: 554715, BD Pharmigen, Franklin Lakes, NJ, USA) according to manufacturer’s instructions. Intracellular IFNγ, IL-2 and TNFα were detected by incubating with anti-IFNγ-APC (Clone XMG1.2, BD Pharmingen, Franklin Lakes, NJ, USA), anti-IL2-PE (Clone JES6-5H4; BD Biosciences, East Rutherford, NJ, USA), and anti-TNFα-AF488 (Clone MP6-XT22; Biolegend) antibodies for 1 h at 4 °C. Cells were washed with a FACS buffer and analysed using a Fortessa X20 (BD Biosciences, East Rutherford, NJ, USA) and analysis performed on FlowJo software V10.8 (TreeStar, Ashland, OR, USA).
4
Flow Cytometry-Based Cellular Profiling
5
Murine CAR T Cell Stimulation Assay
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Murine CAR T cells were cocultured for 16 h in sextuple with one of four stimulation conditions: non‐stimulated (media‐alone), agonistic CD3 plate‐bound antibody (Clone 145‐2C11, BioXcell, Lebanon, USA), 1 × 104 human U87 cells or 1 × 104 human U87WT.EGFRvIIIns GFP‐Luc cells. After 16 h, the cultures were loaded with equivalent numbers of non‐antibody‐binding counting beads (BD, New Jersey Negative Control CompBead Plus), dissociated, collapsed into triplicate wells and transferred to a new plate for antibody labelling. Cells were incubated with either MYC‐tag (GCT02) or FLAG‐tag (C2173) for 1 h on ice. Cells were washed with FACS buffer and labelled with a cell surface murine antibody cocktail for 30 min containing anti‐CD3‐AF700 (BD Pharmingen New Jersey), anti‐CD8‐BV711 (BioLegend, San Diego), anti‐PD‐1 PE‐Cy7 (BioLegend, San Diego), anti‐LAG‐3 (CD223) APC (BD Pharmingen, New Jersey), anti‐CD69 BV786 (BD Horizon, New Jersey) and DAPI (live/dead). The expression of these surface markers on each population was determined by flow cytometry using a BD Fortessa X20 flow cytometer, and data analysis was performed in FlowJo™ software v10 (BD, New Jersey). Where relevant, CAR T cells were directly enumerated by determining cell subset and counting bead numbers, and normalising CAR T cell number to 1 × 104 counting beads to allow comparison across samples.
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