December 21st, 2012
- Wash cells with 1x PBS.
- Suspend cells at 5×10^7 cells/mL in R.T. MACS Buffer with 2.5% PHS (15 mL/500 mL PBS).
- Transfer 8 mL max of cell suspension into a 14 mL round-bottom polystyrene tube.
- Add Anti-Human CD45RO 50 uL/mL and incubate at RT for 15 min.
- Add the Enrichment Cocktail at 50 uL/mL and incubate at RT for 10 min.
- Vigorously pipette Magnetic Nanoparticles and add to cells at 100 uL/mL.
- Mix well and incubate at RT for 10 minutes.
- Bring the cell suspension to a total volume of 10 mL ( > 2×10^8 cells) by adding MACS Buffer.
- Mix the cells in the tube by gently pipetting up and down 3 times.
- Place the tube, without cap, into the magnet.
- Set aside for 10 minutes.
- Carefully and slowly pipet clear cell suspension from the bottom and transfer into a new 14 mL round-bottom polystyrene tube.
- Place the new tube in the magnet for 5 minutes.
- Carefully and slowly pipet clear cell suspension from the bottom and transfer into a new 14 mL conical tube if not labeling with CFSE or a 50 mL conical tube if labeling with CFSE.
- Suspend in 5 mL of 5% PHS RPMI if not labeling with CFSE or 1x PBS if labeling with CFSE.
- Count cells while they spin at 1600 RPM for 5 minutes.
- Bring T cells to 10 x10^6 cells/mL 1x PBS at R.T.
- Add the 10uM diluted stock solution CFSE (dilute factory vial with 18 uL DMSO to obtain 5mM stock; dilute 1:500 for a 10uM diluted stock) at 1:10 to the T cells (100 uL of diluted stock/1 mL of cell suspension.)
- Mix well and incubate 10 minutes at R.T.
- Inactivate the CFSE by washing in 5% PHS at 1600 RPM for 5 minutes.
- Suspend in 5 mL 5% PHS.
- Count cells
- Prepare cell suspension at desired cell concentration.
500 mL 1x PBS
2 mL EDTA
15 mL PHS
5% PHS Media
500 mL RPMI
1.2 mL Gentamycin
5 mL HEPES
Falcon 14 mL Polystyrene Round-Bottom Tubes
BD Biosciences, Catalog # 352057
Silver EasySep Magnet
Stemcell Technologies, Catalog # 19061
CellTrace™ CFSE Cell Proliferation Kit – For Flow Cytometry
Catalog # C34554
Human Naïve CD4+ T cell Enrichment Kit (Negative Selection)
Stemcell Technologies, Catalog # 19155