The set of experiments performed in the assay “20150317-20 PBMCs vs isolated B cells w-w/o stimulation (AF700/PE/Aqua)” have been repeated .
OBJECTIVES
– To compare the flow cytometry results obtained for total PBMCs vs isolated stimulated and non-stimulated B cells, with Monensin. Samples were withdrawn from three individuals.
MATERIALS
– PBMCs and isolated B cells from three individuals (individuals 4,5 & 6 in the database, pre-treatment)
– Isolated B cells using the “EasySep negative selection Human B-cell enrichment kit” (Ref.190549 Stemcell Technologies)
– anti-CD19-AF700 antibody (Ref.302226 BioLegend)
– anti-IL10-PE antibody (Ref. 130-096-043 Miltenyi)
– LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Ref.L34957 Invitrogen)
PROTOCOLS
Note: both PMBCs and purified B cells were analized using fmo (for this purpose all samples were permeabilized). No beads or IC’s were assayed in this experiment.
For PBMCs the following conditions were assayed: non-stained cells, Aqua, Aqua+AF700, Aqua+ PE, AF700 + PE and Aqua+AF700+PE. For 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:
20/04/15 PROTOCOL FOR PBMCs ANALYSIS W/O STIMULATION
As described in the experiment cited above.
Individual 4 (CPP): 20 million cells in 2,5 mL. Of those, 600 µL (6 million cells) were used for PBMCs flow cytometry and the rest were used for B cell isolation and stimulation previous flow cytometry.
Individual 5, (IPP): 20 million cells in 2,5 mL. Of those, 600 µL (6 million cells) were used for PBMCs flow cytometry and the rest were used for B cell isolation and stimulation previous flow cytometry.
Individual 6, (JMGV): 23 million cells in 2,5 mL. Of those, 522 µL (6 million cells) were used for PBMCs flow cytometry and the rest were used for B cell isolation and stimulation previous flow cytometry.
23/03/15 PROTOCOL FOR B CELL ANALYSIS WITH STIMULATION
As described in the experiment cited above.
A) B cell isolation
Individual 4 (CPP): since 6 million cells were used for the PBMCs analysis, our starting material was a 1,4 mL suspension with 14 million cells which was was concentrated to 0,5 mL in order to isolate B cells using the kit described above. After the purification step, 1·106 cells in the 2,5 mL suspension were found.
Individual 5, (IPP): since 6 million cells were used for the PBMCs analysis, our starting material was a 1,4 mL suspension with 14 million cells which was was concentrated to 0,5 mL in order to isolate B cells using the kit described above. After the purification step, 0,625·106 cells in the 2,5 mL suspension were found.
Individual 6, (JMGV): since 6 million cells were used for the PBMCs analysis, our starting material was a 1,5 mL suspension with 17 million cells which was was concentrated to 0,5 mL in order to isolate B cells using the kit described above. After the purification step, 1,2·106 cells in the 2,5 mL suspension were found.
B) Bcell culture and stimulation
Two conditions were assayed in the experiment (stimulated and non-stimulated cells ), so, for all individuals, the 2,5 mL suspension was divided in 2 tubes containing 1,25 mL with:
Individual 4 (CPP): 0,5 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 200 mcL medium.
Individual 5 (IPP): 0,31 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 125 mcL medium.
Individual 6 (JMGV): 0,6 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 240 mcL medium.
In all cases, cells were cultured in 5 mL (12 x 75 mm) polystyrene tubes. 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 withdrawn from culture, sample supernantants were stored at -80ºC.
Flow cytometry was performed as described in previous experiments.
RESULTS
