Preparation of low density, collagenase-digested splenocytes Protocol

1. Dilute 2 ml of 4000 U/ml Collagenase D as follows: 1 ml into 9 ml HBSS/Ca²⁺/Mg²⁺ (=400U/ml) and 1 ml into 39 ml HBSS/Ca²⁺/Mg²⁺ (=100U/ml). Put on ice.

2. Place 5 ml of the 100U/ml solution in a 10 cm dish. Over a dry dish, inject spleens with 100U/ml solution. Hold spleen with forceps and push onto 22-G needle while slowly injecting 1 ml collagenase solution. Tear open the spleen with the needle and transfer to dish with collagenase. After injecting all the spleens, collect any fluid from the dish and put into 50 ml tube. Rinse dish with another 3 ml 100U/ml collagenase and also add to tube.

3. Using forceps, tease the spleens apart into small fragments. Then pipette vigorously with a 5 ml pipette. Tilt the dish, and leaving the larger fragments behind, transfer solution to tube.

4. Add 10 ml 400U/ml collagenase to dish with leftover fragments and incubate at 37oC for 60-90 minutes. Then pipette fragments vigorously and mush with glass slides. Rinse slides with 3 ml 100U/ml collagenase and collect suspension into tube. Rinse dish one last time with 3 ml 100U/ml collagenase and add to tube.

5. Spin, 10 min, speed 3 (ca. 280 g). Resuspend in 1 ml dense BSA per spleen. Overlay up to 5-6 ml splenic suspension in 15 ml conical with 1.5 ml RPMI with no additions. Spin, 15 min, 7000 RPM, HS-4 rotor, 4oC, with slow acceleration and no brake.

6. Take interface plus RPMI and transfer to 15 ml conical. Fill tube with RPMI with no additions and mix. Spin and then do one wash.

7. Resuspend in 5 ml RPMI with no additions. Add 0.25 ml mitomycin C at 0.5 mg/ml. Incubate 37oC, 30 min, then wash 3X.