The staining method used for the detection of RBD and Spike memory B cells has been described previously.32 (link) In brief, biotinylated RBD and Spike proteins were incubated with Streptavidin-PE (SA-PE; Molecular probes; ThermoFisher Scientific) and Streptavidin-APC (SA-APC; BD Pharmingen) in a molar ratio of 4:1 and 2:1, respectively. Cryopreserved PBMCs were thawed rapidly in a 37°C water bath and washed with pre-warmed RPMI media supplemented with 2 mM L-glutamine, 50 IU/mL penicillin, 50 μg/mL streptomycin, and 10% heat-inactivated fetal calf serum (Sigma) then washed twice. A maximum of 1 × 107 cells were stained with Fixable Viability Stain 700 (FVS700) (BD Bioscience in a 1:1000 dilution), 5 μL Human Fc block (BD Bioscience) per 2 × 106 cells, 1 μg/mL each of RBD and Spike tetramers, 5 μL each of CD21 BV421, IgD BV510, CD10 BV605, CD19 BV711 and CD20 APC-H7, 8 μL of IgG BV786, 2 μL each of CD27 PE-CF594 and CD38 PE-Cy7, 2.5 μL HLA-DR BB515 and 0.5 μL CD3 BB700 (BD Bioscience). Cells were washed, resuspended in FACS wash buffer and the data acquired on BD FACSAria™ III. Data analysis was performed using FlowJo version 10.7.1 (TreeStar). To establish background B cell levels for each of antigens and phenotypes we performed the same analysis with PBMCs from 9 healthy control individuals. Background was calculated as: average value + 2SD.
Fixable viability stain 700 fvs700
Manufactured by BD
Sourced in United States
FVS700 is a fluorescent dye used for live/dead cell discrimination. It is designed to measure cell viability in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3, by BD (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Human fc block, by BD (1 mentions)
Cd3 bb700, by BD (1 mentions)
Hla dr bb515, by BD (1 mentions)
Pkh26 dye, by Merck Group (1 mentions)
Cd3 pe cy7, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Facsaria fusion, by BD (1 mentions)
Cd69 bv605, by BD (1 mentions)
Cd62l apc, by BD (1 mentions)
Cd103 pe, by BD (1 mentions)
Cd44 bv421, by BD (1 mentions)
4 protocols using fixable viability stain 700 fvs700
1
Detecting RBD and Spike Memory B Cells
2
Cytotoxicity Assay of B16-OVA Cells
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B16-OVA melanoma cancer cells were seeded in a 96-well plate (30,000 cells per well) in 200 µl of RPMI 10%FBS 1%PSA medium after cell trace™ violet labeling (Invitrogen - Thermo Fischer Scientific). Splenic CD8+ T cells were isolated from OT-I female mice using the CD8a+ T-cell Isolation Kit and cocultured with B16-OVA cells at different ratios of B16-OVA : CD8+ T cells) and treated, or not, with 5 µM of H89. After 48 h of coculture, B16-OVA cell viability was analyzed by flow cytometry using a Fixable Viability stain 700 (FVS 700) (BD Biosciences) and using the cell trace™ violet labeling to differentiate B16-OVA cells from CD8+ T cells.
3
Tracking Mesenchymal Stem Cells in Lungs
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MSCs were labelled with PKH26 dye, according to the manufacturer's protocol (Sigma). Briefly, cell pellets were resuspended in 1 mL diluent C. Separately, 1 mL diluent C was mixed with 4 μL PKH26. The cell suspension was mixed with the stain solution and incubated at room temperature. The labelling reaction was stopped by adding an equal volume of FBS. Labelled cells were centrifuged and washed twice with PBS. One or three days after infusion of PKH26‐stained MSCs, total lung cells were obtained. Then, they were labelled with Fixable Viability Stain 700 (FVS700) (BD), anti‐CD45‐PerCP‐Cy5.5 and anti‐F4/80‐BV421. The live cells were sorted after gating for viability (negative for FVS700), morphology (FSC‐H vs. FSC‐A), expression of CD45, F4/80 and PKH26 dye.
4
Lung Cell Isolation and Flow Cytometry
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An amount of 2 μg of anti-mouse CD45-BV786 antibody (BD Biosciences, Franklin Lakes, NJ, USA) was diluted in sterile PBS and administered intravenously in the tail vein 10 min before anesthesia. CD 45+ is a circulating lymphocyte, and CD45− is a tissue-resident cell. In total, 40 U/mL DNase I and 1 mg/mL Collagenase IV enzyme were measured to a volume of 2 mL and incubated with lung tissue at 37 °C for 30 min. The tissue was then homogenized using a dissociator, filtered through a 70 μM cell strainer, and subjected to RBC lysis to obtain single lung cells. After preparing the lung cells to a concentration of 5 × 106 cell/mL, they were washed with PBS and live-cell staining was performed using fixable viability stain 700 (FVS700) (BD Biosciences, Franklin Lakes, NJ, USA). After treating lung cells with Fc BlockTM reagent, the surface markers CD44-BV421, CD69-BV605, CD103-PE, CD62L-APC, CD4-FITC, CD3-PE-Cy7, and CD8-BB700-per cycle 5.5 ((BD Biosciences, Franklin Lakes, NJ, USA)) were used for staining. The flow cytometry gating strategy was performed according to Supplementary Materials Figure S1 , based on a previous study [45 (link)]. FACS Aria Fusion from BD Biosciences was used, and all results were derived using FlowJo software v10.
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