Stock Solutions#
- FACS Buffer [5590uL per experiment]
- CD90-APC Antibody [10 uL]
- VIOLET L/D [0.5uL]
- IC Fixation Buffer [200 uL]
- Permeabilization Buffer 10x [1 mL] or 1x [4.198 mL]
- cTnT FITC [2 uL]
- PBS
- Distilled Water
Preparation#
- Detach your CMs and count them
- Divide your CMs into 2 eppendorfs (250-300k cells/eppendorf):
- 1st is UNSTAINED - 2nd is STAINED cells
- Centrifuge your cells 2.0 rpm 4min and remove the supernatant
- Add 500uL FACS BUFFER (EDTA 0.5 1:1000, FBS 0.5% in PBS) and centrifuge again
- Remove supernatant
- Add 100 uL FACS BUFFER to UNSTAINED eppendorf
- add 90uL to FACS BUFFER+10uL CD90-APC antibody to STAINED eppendorf
- Incubate on ice 30’ in the dark
- During incubation prepare L/D by adding 0.5uL of VIOLET L/D staining in 500uL of PBS (put on ice)
- At the end of CD90 incubation, add 600uL PBS to the eppendorfs containing your UNSTAINED and STAINEDED cells and centrifuge
- Remove supernatant
- Add 100uL PBS to UNSTAINED
- Add 100uL of L/D solution to STAINED
- Incubate on ice for 30’ in the dark
- At the end of incubation, add 600uL of PBS and centrifuge
- Remove supernatant and add 100uL of IC Fixation buffer to UNSTAINED and STAINED
- Incubate RT for 20 min in the dark
- Prepare Permeabilization Buffer 1X duiluting 1mL of Permeabilization Buffer 10X in 9mL of ultrapure distilled water
- At the end of incubation, add to your UNSTAINED and STAINED eppendorfs 600uL of Permeabilization Buffer 1X and centrifuge
- Remove supernatant
- Add 100uL of Permeabiization buffer 1X to UNSTAINED
- Add 98uL of Permeabilization Buffer 1X and 2uL of cTnT-FITC to STAINED
- Incubate 30’ RT dark
- Go to switch on the FACS and take FACS tube to transfer your samples
- Add to each FACS tube 2mL of PERMEABILIZATION BUFFER 1X
- At the end of incubation, transfer your cells in the relative FACS tube and centrifuge
- Remove supernatant and add 2mL/tube of FACS buffer
- Centrifuge, remove supernatant, and add 200uL of FACS buffer in each tube and go to read your results to FACS