IL-10 production by non-stimulated and stimulated B cells will be assessed using the “IL-10 Secretion Assay – Detection Kit (PE), human ” kit by Miltenyi instead of our usual FC protocol, as in the 14/08/15 experiment. The only difference relies in using FBS-free medium w/o stimuli during the 45 min. incubation step.
OBJECTIVES
– To compare the flow cytometry results obtained for total PBMCs vs isolated stimulated and non-stimulated B cells. Samples were withdrawn from individuals 50 PRE in the new database.
MATERIALS
– PBMCs and isolated B cells from two individuals: individual 50 (GGP) PRE
– “EasySep negative selection Human B-cell enrichment kit” (Ref.190549 Stemcell Technologies)
-“IL-10 Secretion Assay – Detection Kit (PE), human ” kit Ref: 130-090-434, Miltenyi.
– anti-CD19-AF700 antibody (Ref.302226 BioLegend)
– LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Ref.L34957 Invitrogen)
PROTOCOLS
Prior to PBMCs isolation,1 out of the 5 heparin sample tubes per individual were centrifuged 10 min at 1500 rpm in order to obtain plasma. 1 mL of plasma per sample were aliquoted and stored at -80ºC. Samples were reconstituted to their original volume with PBS and processed as per usual.
Note: both PMBCs and purified B cells were analized using fmo. No beads or IC’s were assayed in this experiment.
For both PBMCs and isolated B cells only the Aqua+AF700+PE condition was assayed. Both assays were conducted using the same settings as in previous experiments.
2 different assays are included in this protocol:
25/04/16 PROTOCOL FOR PBMCs ANALYSIS W/O STIMULATION
A) PBMCs isolation
Density gradient centrifugation using Ficoll was performed as described in previous experiments.
Individual 50 PRE: 170.7 million cells in 3 mL. Of those, 18 µL (1 million cells) were used for PBMCs flow cytometry and the rest were used for B cell isolation and stimulation previous flow cytometry.
B) IL-10 secretion analysis by FC
1. Use one million total cells in a 2 ml closable tube per sample.
Note: For larger cell numbers, scale up all volumes accordingly. For less than one million cells, use same volumes.
2. Wash cells by adding 1-2 ml of cold buffer, centrifuge at 300xg for
10 minutes at 4-8°C, pipette off supernatant completely.
Note: Do not remove supernatant by decanting. This will lead to cell loss and incorrect incubation volumes.
3. Resuspend cell pellet in 90 μl of cold medium per million total
cells.
4. Add 10 μl of IL-10 Catch Reagent per million total cells, mix well
and incubate for 5 minutes on ice.
5. Add warm (37°C) FBS-free medium w/o stimuli to dilute the cells accoding to the following proportions.
– If less than 5% IL-10 secreting cells are expected: add 1 mL of warm medium per million cells. (our case)
– If more than 5% IL-10 secreting cells are expected: add 10 mL of warm medium per million cells.
6. Incubate cells in closed tube for 45 minutes at 37°C under slow
continuous rotation or turn tube every 5 minutes to resuspend settled cells.
7. Put the tube on ice.
8. Wash the cells by filling up the tube with cold buffer, centrifuge
at 300xg for 10 minutes at 4-8°C. Pipette off supernatant
completely.
Note: If the volume of the cell suspension was higher than the volume of added buffer, repeat wash step.
9. Resuspend cell pellet in 90 μl of cold buffer per million cells.
10. Add 10 μl of IL-10 Detection Antibody (PE) per million
cells.
10. Add additional staining reagents: 2 µL of the CD19-AF700 antibody per million cells.
11. Mix well and incubate for 10 minutes on ice.
12. Wash cells by adding 2 ml of cold buffer, centrifuge at 300xg for
10 minutes at 4-8°C, pipette off supernatant.
13. Add 1 mcL of the reconstituted fluorescent reactive dye (Aqua Live/Dead Stain) per million cells to 1 mL of the cell suspension and mix well. Incubate at room temperature or on ice for 10 minutes, protected from light.
14. Proceed to analysis. Acquire 200.000 viable cells from each sample.
27/03/16 PROTOCOL FOR B CELL ANALYSIS AFTER STIMULATION
A) B cell isolation
Individual 50 PRE: since 1 million cells were used for the PBMCs analysis, our starting material was a 2.982 mL suspension with 169.7 million cells which was concentrated to a final volume of 3.39 mL in order to isolate B cells using the kit described above. After the purification step, 1.85 million cells in the 2 mL suspension* were found.
*there were 2 mL left due to an accidental spill.
B) Bcell culture and stimulation
Two conditions were assayed in the experiment (stimulated and non-stimulated cells ), so, for all individuals, the 2 mL suspension was divided in 2 tubes containing 1 mL with:.
Individual 50 PRE : 0.925 million cells each . To bring the concentration to 2.5·106 cells/ml, each eppendorf was centrifuged at 300g 5 min and the pellet was resuspended in 370 mcL medium.
In all cases, cells were cultured in 5 mL (12 x 75 mm) polystyrene tubes w/o adding Monensin. Since those were same tubes in which the following staining and flow cytometry analysis were performed, and thus no cell loss was expected, after 48h no cell counting was conducted. Once cells were harvested from culture:
– 1×100 mcL (1×250.000 cells) aliquote of patient’s 50 sample was pelleted and stored in RNase free microtubes at -80 ºC.
– culture medium supernantants were stored at -80ºC.
C) IL-10 secretion analysis by FC
The same steps described above were followed.
RESULTS
