Dec 15, 2023
Flow cytometry for ex vivo stimulated pMacs
- 1University of Florida
Protocol Citation: Rebecca Wallings 2023. Flow cytometry for ex vivo stimulated pMacs. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx9x4gx1/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: 92326
Funders Acknowledgement:
ASAP
Grant ID: ASAP-020527
Abstract
Flow cytometry for ex vivo stimulated pMacs
Incubate cells for 30 minutes at 37 degrees in incubator in media containing 3mM EDTA at pH 6.2 to gently lift cells from plate. To encourage dissociation from plate, gently tap plate, or alternatively use a mini scraper
Aliquot cells in to wells of v-bottom 96 well plate
Spin cells at 300 x g at 4 degrees for 5 minutes
Resuspend cells in 50uL of antibody cocktail containing live/dead dye and Fc block
Incubate at 4 degrees in the dark for 20 minutes
Spin cells at 300 x g at 4 degrees for 5 minutes
Remove supernatant and resuspend pellet in 200uL 1 x PBS
Spin cells at 300 x g at 4 degrees for 5 minutes
Remove supernatant and resuspend pellet in 200uL 1 x PBS
Spin cells at 300 x g at 4 degrees for 5 minutes
Remove supernatant and resuspend pellet in 50uL ICC fixation buffer (Invitrogen)
Incubate cells at room temperature for 10 minutes in the dark
Spin cells at 300 x g at 4 degrees for 5 minutes
Remove supernatant and resuspend pellet in 200uL FACS buffer and proceed to assess cells on cytometer