Dec 15, 2023
Patient PBMC flow cytometry
- 1University of Florida
Protocol Citation: rebeccawallings 2023. Patient PBMC flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm688l5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 14, 2023
Last Modified: December 15, 2023
Protocol Integer ID: 92335
Funders Acknowledgement:
ASAP
Grant ID: ASAP-020527
Abstract
Patient PBMC flow cytometry
1 x 106 PBMCs were taken for flow cytometry and transferred to a v-bottom 96-well plate
(Sigma, CLS3896-48EA) and centrifuged at 300x g for 5 minutes at 4°C.
Cells were resuspended in 50 µL of PBS containing diluted fluorophore-conjugated antibodies and incubated in the dark at 4°C for 20 minutes. Cells were centrifuged at 300x g for 5 minutes at
4°C and washed in PBS x 2. Cells were fixed in 50 µL of 1% paraformaldehyde (PFA) at 4°C in the dark for 30 minutes.
Cells were centrifuged at 300x g for 5 minutes and resuspended in 200 µL FACs buffer (PBS, 0.5 mM EDTA, 0.1% sodium azide).
Cells were taken for flow cytometry on a MACS Quant Analyzer (Miltenyi). A minimum of 100,000 events were captured per sample and data were analyzed using FlowJo version 10.6.2 software (BD Biosciences).
When validating flow cytometry panels and antibodies, fluorescence minus one controls (FMOCs) were used to set gates and isotype controls were used to ensure antibody-specific binding.