OBJECTIVE

1. To evaluate the amount of B cells producing IL10
2. To analyze if they all are plasma cells as previously reported
3. To compare 2 different IL10 antibodies and see if results are consistent

MATERIALS

•  PBMCs (different donors)
• Antibodies: CD19-FITC, CD138 PE, IL10 PE, IL10 APC. Isotype controls.
• BD cytofix/cytoperm
• PBS/BSA 2%
• PFA 4% in PBS
• MAQSQuant cytometer

PROCEDURE

We followed the same procedure described before. The only change was the addition of a new set of antibodies: CD138-PE and IL10-PE, so compensation was re-done. In subsequent days, considering the discrepance in signal between the IL10-APC and the IL10-PE we did other confirmatory experiments. We also tested same samples letting the cells sit overnight to see if there were any changes due to the prolonged time of cytometry analysis. Results explained below

RESULTS

– IL10+ B cells in healthy donors, tested by IL10-APC are around 0.2-0.7% of the B cells, BUT, IL10-PE does not work and, adding the IL10-PE antibody and redoing the compensation damps the IL10-APC signal. See later

  – When analyzing the same samples showed before, but applying the voltages and comensation set-up done in experiments without the B2 channel (in those that we did not use PE antibodies), the IL10-APC positive cells appear much clearer. Something’s wrong then with the compensation set up in the CD 19IL10 FITC/(APC-PE) experiment.

– This problem only happens when the IL10PE antibody is used. When we did the same experiment, adding CD138-PE (with same voltages and same compensation setup), the IL10-APC cells were perfectly shown (see below). That same experiment showed us no CD138+ cells among the CD19+IL10+ population.

– There’s no big difference in doing experiments one day and analyzing the next one than doing eveything the same day (see first image – 0H – and the one below -24H-)

CONCLUSIONS

– B cell purification works very well. More than 95% of all purified cells are CD19+

– We have to confirm that the numbers of IL10 positive cells seen with the IL10APC antibody are true. There’s no doubt that the staining is different in IC than in IL10 antibody, but i think we have to confirm with an independent IL10 antibody. So far, the IL10PE antibody is not working but, probably is due to the use of the compensation set-up from the CD138PE antibody. If we repeat these experiments we should have a nice positive control and test both antibodies in parallel.

– CD138+ cells are scarce within the CD19 population. There are some, but they are not IL10 positive. I don’t think we have to lose much time with this, although, if we are able to confirm it, it could be very interesting.