CD4 enrichment using AutoMACS Abs
Reagents are located in 2 boxes in the mouse tissue culture room fridge.
Labeled “mouse CD4 enrichment 1o mAbs, SAB bio abs.”
And “ mouse CD4 enrichment 2o mAbs and beads, Stem Sep antibiotin TAC (13050C) and Stem sep magnetic colloid (10051), ”
Magnets are in Bay 3, 3rd drawer down next to Amy’s fridge, or above Meli’s bench.
Protocol
1. Obtain single cell suspensions from mice.
2. Resuspend cells at 108 cells/ml in 2%FCS - HBSS buffer (staining and rinsing buffer)
3. Ab incubations can be done in the 15mL Falcon tubes, but make sure that cells are then transferred to polystyrene tubes (it can be done in the washing process after the first set of Ab staining)
4. Add the following cocktail of 1o biotinylated antibodies :
CD117 (1:200)
Ly6G, MHC class II, CD19, CD24, CD8 (1:400)
5. Incubate at 4°C for 15min. Best results in fridge not on ice.
6. Wash 1X in rinsing buffer, spin at 1300rpm for 10min.
7. Add anti-biotin tetrameric antibody (1:10 dilution) or 20uL Ab / 200uL of cells. Mix well.
8. Incubate at 4°C (fridge) for 15min.
9. Vortex the magnetic colloid and add 1:33 dilution (6uL Ab / 200uL). Mix well.
10. Incubate at 4°C (fridge) for 15min.
11. Prepare polystyrene tube in magnet of appropriate size : up to 2.5ml small, 2.5 – 5ml large.
bring the cell suspension to a total volume up to 2.5mL or 5mL.
12. Add cells to the tube in the magnet and Incubate for 5 min at RT.
13. Using the 2mL or 5mL pipette (small / large magnets respectively) pipette out slowly the negative fraction of CD4+ cells making sure not to touch the sides of the tube.
14. Filter and wash.
*** CD4 purity ranges between 55-74%