OBJECTIVES
– To test whether the new ODN vial recevived 20170123 properly stimulates B cells. We doubt its viability due to its temperature upon arrival, hence this FC test following our breg fc protocol.
MATERIALS
– isolated B cells from one individual: individual ct ana
– “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
Note: B cells were analized using fmo. No beads or IC’s were assayed in this experiment. Only the Aqua+AF700+PE condition was assayed. This assay was conducted using the same settings as in previous experiments.
A) PBMCs isolation
PBMCs were isolated using CPT tubes as explained in previous experiments. 24.3 million cells in 2 mL, all of them were used for B cell isolation and stimulation previous flow cytometry.
B) B cell isolation
Our starting material was a 2 mL suspension with 24.3 million cells which was concentrated to a final volume of 0.500 mL in order to isolate B cells using the kit described above. After the purification step, 1.875 million cells in the 2,5 mL suspension were found.
B) Bcell culture and stimulation
Three conditions were assayed in the experiment (ODN only stimulated B cells, ODN+anti G/M/A Ab stimulated B cells and non-stimulated cells ), so, for all individuals, the 2,5 mL suspension was divided in 8 tubes containing 0,83 mL with:.
Individual 55 EBL POST : 0.6445 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 258 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:
C) IL-10 secretion analysis by FC
Once cells were harvested from culture:
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.
RESULTS
