OBJECTIVE
To sort plasmablasts, naive B cells and memory B cells to see if we can detect antibodies against CNTN1/CASPR1 complex in these subsets.
MATERIALS
Same procedure as in the setting-up experiment
PROCEDURE
- Transfer all blood to 2x50mL falcons and spin down cells at 900g 10′
- Remove plasma and store it frozen
- Add RPMI (w/o additives) up to 50mL again and re-suspend.
- Prepare 4 x50mL Falcons with 15mL Lymphoprep each.
- Add 25mL of cell suspension to each Falcon w/ Ficoll and spin at 400g 20′ w/o brake
- Get PBMCs and re-suspend in RPMI (the interphase of PBMCs is blurry, the same way it was in the last sorting experiment). Spin down at 300g to wash (2x)
- Re-suspend in 5mL and count (counted 100xmill cells total, aprox)
- Prepare these tubes:
Tube 1: CD19-FITC only
Tube 2: CD27-PerCPCy5.5 only
Tube 3: CD38-PE only
Tube 4: Only cells
Tube 5: All antibodies
- All compensation tubes 1.5 mill cells (in 1mL)
- The rest of cells (120 mill aprox.) into 3mL of RPMI for sorting
- Added 5uL of each antibody for compensation and 100uL for the sorting tube
- Incubate Abs 30min in the dark
- Wash 2x with RPMI
- Prepare 3 tubes (CD19+CD27+CD38+, CD19+CD27+CD38-, CD19+CD27-CD38-) with basal medium
- Go to sort
- After sorting spin down cells at 400g and re-suspend in 100uL of basal medium plus IL6 (1ngr/ml) and count
- Plate 100uL with all sorted cells from each subset in a 96-well plate with basal medium + IL6. Wait one week to collect supernatant.
RESULTS
- Sample was dirty. Not as dirty as in previous experiment. Many red cells but much less than in previous experiment.
- Few CD27+ cells but more than in LAC. It can be possible that the patient does not have many CD27+ B cells because of the treatment (high doses of steroids and IVIg). We’ll know better after doing regular FACS tomorrow with the same sample.
- Plasmablast population was unfrequent but we could isolate approximately 1300 plasmablasts.
- The other two subsets had enough cells to culture (see reports below)
Global Sheet1_20022013132246 Sort_Report_20022013132204
CONCLUSIONS
Regular FACS analysis pending to see the actual proportions of each population.
