The set of experiments performed in the assay “20150225-27 PBMCs vs (non-)stimulated B cells (AF700/PE)” have been repeated with the addition of the Aqua viability stain.
OBJECTIVES
– To compare the flow cytometry results obtained for total PBMCs vs isolated B cells stimulated and non-stimulated, with Monensin. Samples were withdrawn from one control (Sonia).
MATERIALS
– PBMCs and isolated B cells from one control (Sonia)
– 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 each sample the following conditions were assayed: non-stained cells**, Aqua, Aqua+AF700, Aqua+ PE, AF700 + PE** and Aqua+AF700+PE.
** These samples contain Aqua stain by mistake.
2 different assays are included in this protocol:
05/03/15 PROTOCOL FOR PBMCs ANALYSIS W/O STIMULATION
1) Dilute blood 1: 1 with SF and mix by inversion. PBS without Ca and Mg or medium may also be used.
2) In a 15 ml tube, add 4 mL of Ficoll. Add dropwise sliding through the tube wall 6 mL of diluted blood with a syringe which has been stripped of the plunger. Use a 1,1×25 mm needle (yellow).
If the tube is large (50 mL), add 15 mL of Ficoll and 25 mL of blood, decanting the blood tube and adding dropwise with a pipette.
3) Centrifuge 30 min at 400g at RT without brake (remove acceleration and deceleration).
4) Using a glass-made Pasteur and a pear (be careful not to squeeze inside the tube) draw the cloud of leukocytes in the sample and placed it in a 50 mL Falcon containing 10 mL of SF. Fill Falcon with SF to approx. 45 mL.
5) Centrifuge at 300g for 5 min with brake and acceleration at RT and remove supernatant without touching the pellet.
6) Resuspend the pellet by adding to 40 mL of SF.
7) Centrifuge at 300g for 5 min with brake and acceleration at RT and remove supernatant without touching the pellet.
8) Resuspend the pellet in 2 ml in staining buffer. (PBS + 1% FCS)*
* 50 mL: 500 µL FCS + PBS w/o Ca or Mg.
9) Perform cell count.In this assay, the samples had:
Sonia: 27 million cells in 2 mL. Of those, 444 µL (6 million cells) were used for PBMCs flow cytometry and the rest were used for B cell isolation and stimulation previous flow cytometry.
Note: from this point on always work with the samples on ice.
11) Resuspend the pellet by adding enough mL of staining buffer to each Falcon as to reach the desired cell concentration Adjust the density with PBS to 1 × 10^6 cells in a 1 mL volume for each test condition.
In this assay 6 million cells per each assayed individual were resuspended in 6 mL of staining buffer, so each eppendorf cantained 1 million cells.
12) Add 1 mcL of the reconstituted Aqua fluorescent reactive dye to 1 mL of the cell suspension and mix well.
13) Incubate at room temperature or on ice for 30 minutes, protected from light.
14) Centrifuge for 5 min at 400 g at 4 ºC and remove supernatant.
15) Wash the cells with 1 mL of staining buffer.
16) Add staining buffer and incubate 30 min approx. Ratio: 100 mL of staining buffer per million cells.
17) Add 2 uL of the AF700 antibody per million cells. Incubate 30 min at 4 ° C (ice).
18) Skip beads preparation (no beads were used in this assay).
19) Add 500-600 mL of staining buffer to the microtubes obtained in step 15.
20) Centrifuge for 5 min at 400 g 4 ºC and remove supernatant.
21) Add 1 mL of blocking solution and repeat step 18.
22) Permeabilization ( protocol for permeabilization conducted at a later time)
22.1. Remove the supernatant and add 100 µL of a 4% paraformaldehyde in PBS solution to the microtubes . Incubate 10-20 min at 4 ° C.
22.2 Centrifuge for 5 min at 400 g 4 ºC and remove supernatant.
22.3 Add 500-600 µL of staining buffer and centrifuge for 5 min at 400 g 4 ºC
22.4 Remove supernatant and repeat step 22.3
22. 5 Remove supernatant and resuspend cells in 500 µL staining buffer for storing cells at 4ºC for up to 72h (left overnight only).
22.6 Centrifuge for 5 min at 400 g 4 ºC and remove supernatant.
22.7 Add 250 uL of PermWash solution and incubate 15 min.
22.8 Centrifuge for 5 min at 400 g 4 ºC and remove supernatant.
22.9. Add 100 uL of PermWash solution plus 10 µL of PE antibody per million cells in the microtube.
22.10. Incubate 30 min at 4 ° C.
22.11 Centrifuge 5 min at 400 g at 4 ° C. Discard the supernatant and wash with 250 mL of PermWash solution.
22.12. Discard the supernatant and wash again with 250 mL of PermWash.
22.13. Discard the supernatant and resuspend in staining buffer.
06/03/15 PROTOCOL FOR B CELL ANALYSIS WITH STIMULATION
After the previously described steps 1-9:
A) Bcell isolation using “EasySep negative selection Human B-cell enrichment kit” by Stemcell Technologies (#190549) (Manual protocol using the purple easysep magnet)
1)Prepare cell suspension (500 μL – 2 mL) at a concentration of 5 x 107 cells/mL (up to 1 x 108 cells) in the recommended solution (PBS + 2% FBS + 1 mM EDTA.). Cells must be placed in a 5 mL (12 x 75 mm) polystyrene tube* to properly fit into the Purple EasySep Magnet.
Sonia: since 6 million cells were used for the PBMCs analysis, our starting material was a 2 mL suspension with 20 million cells which was was concentrated to 500 mcL by centrifugation at 300g for 5 min with brake and acceleration at RT.
2) Add the Human B Cell Enrichment Cocktail at 50 μL/mL of cells (e.g. for 2 mL of cells, add 100 μL of cocktail). Mix well and incubate at room temperature for 10 minutes.
3) Vortex the D Magnetic Particles for 30 seconds. Ensure that the particles are in a uniform suspension with no visible aggregates.
4) Add the D Magnetic Particles at 75 μL/mL of cells (e.g. for 2 mL of cells, add 150 μL of magnetic particles). Mix well (Careful: place tap in order to vortex aoutside the flux cabin) and incubate at room temperature for 5 minutes.
5)Bring the cell suspension up to a total volume of 2,5 mL by adding recommended solution. Mix the cells in the tube by gently pipetting up and down 2 – 3 times.
6) Place the tube (without cap) into the magnet. Set aside for 5 minutes.
7) Pick up the magnet, and in one continuous motion invert the magnet and tube, pouring off the desired fraction into a new 5 mL polystyrene tube. Leave the magnet and tube in inverted position for 2 – 3 seconds, then return to upright position.
Caution: do not shake or blot off any drops that may remain hanging from the mouth of the tube.
8) Perform cell count as described in previous steps.
Sonia: 1·106 cells in the 2,5 mL suspension were found.
B) Bcell culture and stimulation
1) Culture B cells at a density of 2,5·106 cells/ml in complete medium: RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin.
Two conditions were assayed in the experiment (stimulated and non-stimulated cells ), so the 2,5 mL suspension was divided in 2 eppendorfs containing 1.25 mL with 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 of medium. Cells were cultured in 96-well plates.
2) Stimulate due B cells with 6 µg/ml CpG-B 2006 and 10 µg /ml Goat Anti-Human IgA + IgG + IgM for 48 h at 37°C, 5% CO2.
3) After 42h add, in all conditions, 4 µL of BD GolgiStop (monensin)* for every 6 mL of cell culture and incubate at 37°C, 5% CO2 for 5-6h.
4) After this time, harvest cells in a sterile eppendorf. Clean the well with additional 500 µL culture medium and add them to the cell suspension. Centrifuge for 5 min at 400 g at 4 ºC and remove supernatant.Resuspend the pellet by adding staining buffer PBS+ FCS 1%* pH 7.4-7.6.
5) Perform cell count as described in previous steps.
Sonia: 100.000 stimulated and 75.000 non-stimulated cells were recovered from their respective wells.
Proceed with the flow cytometry protocol previously described for the PBMCs analysis.
RESULTS
A) FOR PBMCs ANALYSIS W/O STIMULATION (05/03/2015)
B) FOR B CELLS ANALYSIS W-W/O STIMULATION (06/03/2015)
Repeating the analysis for the suposedly plasma cell subset:
CONCLUSIONS
– When freshly analyzing PBMCs, mortality yields are remarkably low (99,7% viability) comparable to those obtained while culturing B cells with stimulation (98% viability). However, when culturing with a lack of stimuli, B cell viability drops to a 54,6%. Although these results must be considered carefully (due to an incubator problem related to CO2), it seems B cells benefit from stimuli in terms of survival.
– B cell stimulation provides higher IL10 rates (0,21%) vs non-stimulated B cells (0,073%) or PBMCs (0,060%). This comes to prove that the stimulation described by Lin et al. 2014 works, despite being Breg a rather scarce subset.
– When working with FlowJo, the original population selection for both PBMCS (selecting Lymphocytes) and B cells (selecting what should be true CD19+ cells) can’t be dragged from one analysis to the other. The same happens for viable cells selection. It might, most likely, be due to a change in cell morphology during culture.
– As a result of the morphology variation, a second layout for what might have been plasma cells is included in the results. This subset might not be the expected one since all of the selected cells are highly CD19+.
– As a consequence of the observed variability in the whole B cell lineage, a trial was made to analyze the entire viable population of purified B cells, not focusing on one particular subset. However, due to the presence of multiple subsets, this analysis cannot be performed. In further experiments we will continue to pick the most remarkable cell subset and gate it with the fmo data obtained from the PBMCs analysis (despite the change in cell morphology).
