Washed PBMCs were resuspended in an appropriate volume of PBS to obtain a cell concentration of 107 cells/mL. Cells were incubated with a viability reagent, Cell-ID Cisplatin (Fluidigm, South San Francisco, CA) at a final concentration of 5 μM for 5 min on ice. Cisplatin was quenched by washing once with 5x volume of MaxPar® Cell Staining Buffer (Fluidigm, South San Francisco, CA) and centrifuged at 300 × g, then resuspended to a final concentration of 30 million cells/mL in staining buffer. To start antibody labeling, 3 million cells were transferred to Falcon® 5 mL 12 × 75 mm tubes (Corning, Corning, NY) and incubated with 5 μL of Human TruStain FcX (BioLegend, San Diego, CA) for 10 min on ice to block Fc receptor binding. Healthy PBMCs were stained with CD45-198Pt (inhouse conjugation of Clone HI30, Biolegend, San Diego, CA) as a reference to incorporate into each tumor sample as our internal reference control and tumor cells were stained with CD45-89Y (Fluidigm, South San Francisco, CA) for 30 min on ice. Cells were washed twice with 4 mL cell staining buffer before being prepared for surface staining.
Falcon 5 ml 12 75 mm tubes
Manufactured by Corning
The Falcon® 5 mL 12 × 75 mm tubes are a laboratory product designed for a variety of applications. They have a capacity of 5 mL and measure 12 × 75 mm in size. The tubes are made from high-quality materials to provide reliable performance in the laboratory setting.
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2 protocols using falcon 5 ml 12 75 mm tubes
1
PBMC Viability Staining and Immunophenotyping
2
PBMC Viability Staining and Immunophenotyping
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Washed PBMCs were resuspended in an appropriate volume of PBS to obtain a cell concentration of 107 cells/mL. Cells were incubated with a viability reagent, Cell-ID Cisplatin (Fluidigm, South San Francisco, CA) at a final concentration of 5 μM for 5 min on ice. Cisplatin was quenched by washing once with 5x volume of MaxPar® Cell Staining Buffer (Fluidigm, South San Francisco, CA) and centrifuged at 300 × g, then resuspended to a final concentration of 30 million cells/mL in staining buffer. To start antibody labeling, 3 million cells were transferred to Falcon® 5 mL 12 × 75 mm tubes (Corning, Corning, NY) and incubated with 5 μL of Human TruStain FcX (BioLegend, San Diego, CA) for 10 min on ice to block Fc receptor binding. Healthy PBMCs were stained with CD45-198Pt (inhouse conjugation of Clone HI30, Biolegend, San Diego, CA) as a reference to incorporate into each tumor sample as our internal reference control and tumor cells were stained with CD45-89Y (Fluidigm, South San Francisco, CA) for 30 min on ice. Cells were washed twice with 4 mL cell staining buffer before being prepared for surface staining.
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