OBJECTIVES
– To compare the CD19+ IL10+ subset in PBMCs of two control samples (Luis vs Xavi).
– To determine whether reducing the concentration of both antibodies (keeping the same ratio as described in previous assays) affects the outcome of the experiment.
PROTOCOL (described in the setup experiment)
1. In this experiment no beads or isotypes control were assayed.
2. Cell count:
- Xavi: 2.5 ·10^6 cells/mL–> 5·10^6 cells in the 2 resuspended mL
- Luis: 2 · 10^6 cells/mL–> 4·10^6 cells in the 2 resuspended mL
3. As PBS was not used in step 8 (blocking solution was used instead), step 10 was not followed and consequently in step 11 there was no pellet to resuspend. In this particular step, enough blocking solution was added to each sample in order to obtain aliquots containing 10^6 cells/mL:
- Xavi (5·10^6 cells/ 2 mL)–> 3 mL were added to the previous 2 mL
- Luis (4·10^6 cells/ 2 mL)–> 2 mL were added to the previous 2 mL
Out of those volumes, only one 1 mL aliquot was used in the assay per control sample. The remaining volumes (Xavi–>4 mL and Luis–>3 mL), were frozen according to the following protocol:
- Centrifuge 5 min at 300 g at RT (with brake and accelaration) and carefully remove supernatant. Leave as little blocking solution as possible.
- Resuspend with 1 mL of freezing medium (fetal bovine serum + DMSO 10%) filtered by 22 um. Add the solution to a labeled cryotube and store at -80ºC/ liquid N2.
4. In step 15, the following volumes of FITC antibody per million cells were added:
- Condition A: 20 uL (recommended volume by the manufacturer)
- Condition B: 10 uL
- Condition C: 5 uL
5. In step 21.4, due to a lack of APC antibody,the following volumes per million cells were added:
- Conditions A and B: 5 uL of Anti-Human IL-10 PE-Cyanine 7 (a change in the cytometer from R1 to B4 lane will be needed).
- Condition C: 3,1 uL of APC antibody.
RESULTS
CONCLUSIONS
– As for the antibody concentration, although no conclusive results can be drawn out of the experiment due to the unexpected lack of the proper anti-IL10 antibody for conditions A and B, it seems that reducing the antibody volume (to 25% of the reccomended one) affects not so much the CD19+ tinction by FITC, but the IL-10 tinction by APC as seen in condition C. In this condition a loss of resolution can be detected.
– However, since samples were not analized fresh after the tinction but were kept overnight at 4ºC, results may be affected by a loss of antibody reactivity too. From this assay onwards, samples will be filtered and freshly assessed in the cytometer right after the permeabilization/tinction step.
