Individual Thymic Stromal Cell Digestion

 

Clean each thymus thoroughly of fat and connective tissue

Nick thymic capsule with scissors

Incubate at 37 degrees for 10mins in 2mL of 0.125% (w/v) Collagenase D with 500uL of 0.1% (w/v) DNAse I (both from Roche, Germany) in RPMI-1640, with regular agitation.

Collect supernatant, and put on ice

Repeat the digestion using the remaining settled thymic fragments

After 3 digestions incubate the remaining aggregates for 10mins in 1mL of 0.125%(w/v) Collagenase/Dispase ( Roche , Germany ) with 0.1%(w/v) DNAse I in RPMI-1640.

Centrifuge cells from all fragments at 472gmax for 5mins and resuspent in EDTA/FACS Buffer

Filter cells through 100um mesh and perform cell counts (Coulter Counter)