OBJECTIVE
To sort plasmablasts, naive B cells and memory B cells to see if we can detect antibodies against NF155 in these subsets.
MATERIALS
Same procedure as in the setting-up experiment
Only difference is that tubes came from Granada overnight and that they used Hep-Li tubes with plastic balls inside. That had to be removed before proceeding with Ficoll.
PROCEDURE
- Transfer all blood to 2 50mL falcons and spin down cells at 900g 10′
- Remove plasma and store it frozen (Box with LAC plasma from Miquel)
- 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. For some strange reason cells are not well separated). 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 1mill cells (in 1mL)
- The rest of cells (90mill aprox.) into 3mL of RPMI for sorting
- Added 5uL of each antibody for compensation and 200uL 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 extremely dirty. Many red cells, many dead cells.
- Very few CD27+ cells. Concentrations of antibodies were the same as in the set-up experiment. Something went wrong with either the sample or the staining. It can be possible that the patient does not have many CD27+ B cells. We’ll know better after doing regular FACS tomorrow with the same sample.
- Plasmablast population was close to zero due to low CD19+CD27+ numbers.
- The other two subsets had enough cells to culture (see reports below)
Global Sheet1_14022013143136 Sort_Report_14022013143055
CONCLUSIONS
We are not going to see anything in the plasmablast subpopulation…unfortunately.
Tomorrow I’ll do regular FACS to analyze B cell subsets and see the percentages of each subset to see if they match with the sorting.