Cell Viability Assessment via Zombie Aqua Staining

Cell viability was determined by Zombie Aqua staining (29 (link)) (Biolegend, San Diego, CA), then incubated with fluorophore-conjugated antibodies at 1:10 dilution for 15 minutes at 4 C, then washed twice and resuspended in 2% PFA solution until analysis. Intracellular flow cytometry was performed (30 (link)) and analyzed using an LSR Fortessa cytometer (BD Biosciences), and FlowJo version 10 software (Ashland, OR). Median fluorescence intensity (MFI) fold change calculated by normalizing after subtracting the isotype control MFI.

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Concha-Benavente F., Srivastava R.M., Trivedi S., Lei Y., Chandran U., Seethala R.R., Freeman G.J, & Ferris R.L. (2015). Identification of the cell-intrinsic and extrinsic pathways downstream of EGFR and IFNγ that induce PD-L1 expression in head and neck cancer. Cancer research, 76(5), 1031-1043.

Publication 2015

Zombie Aqua staining LSR Fortessa cytometer

Antibodies Cell viability Dilution Flow cytometry Fluorescence Intracellular Isotype

Corresponding Organization : UPMC Hillman Cancer Center

Other organizations : University of Michigan–Ann Arbor, Harvard University, Dana-Farber Cancer Institute

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Variable analysis

independent variables

Zombie Aqua staining
Fluorophore-conjugated antibodies

dependent variables

Cell viability
Median fluorescence intensity (MFI) fold change

control variables

Antibody dilution (1:10)
Incubation time (15 minutes)
Incubation temperature (4°C)
Washing procedure (twice)
Resuspension in 2% PFA solution
Flow cytometry analysis using LSR Fortessa cytometer and FlowJo software

positive controls

Isotype control for MFI fold change calculation

negative controls

Not explicitly mentioned

Annotations

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