OBJECTIVES
-To set up a new protocol based on the paper by Lin et al. “Human Regulatory B Cells Combine Phenotypic and Genetic Hallmarks with a Distinct Differentiation Fate” to study the Breg subset in two controls (Noemí and Edu) using total PBMCs.
MATERIALS
– PBMC’s
– anti-CD19-AF700 antibody (Ref.302226 BioLegend) and AF700 Mouse IC (Ref.400144 BioLegend)
– anti-IL10-PE antibody (Ref. 130-096-043 Miltenyi) and PE Rat IC (Ref.130-102-645 Miltenyi )
PROTOCOL
In contrast to other assays, anti-CD19-AF700 and anti-IL10-PE were used. IC’s and beads were also used in the assay.
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).
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)
9) Perform cell count:
Noemí: 3.1 million cel/mL, in 2 mL, 6 million cells.
Edul: 1.3 million cel/mL, in 2 mL, 3 million cells.
11) Add enough mL of staining buffer to each Falcon as to reach the desired cell concentration. (aprox 1 million cells/1 mL for each test condition).
In this assay million cells per each assayed individual were resuspended in 5 mL of staining buffer, so each eppendorf cantained aprox. 1 million cells.
12) Regarding each condition, aliquot 1 mL into a microtube labeled correspondingly.
13) Centrifuge for 5 min at 400 g at 4 ºC and remove supernatant.
14) Add staining buffer and incubate 30 min approx. Ratio: 100 mL of staining buffer per million cells.
15) Add 2 uL of the appropriate antibody (anti-CD19-AF700 or AF700-isotype control, condition depending on the test) per million cells. Incubate 30 min at 4 ° C (ice).
16) Prepare compensation beads for AF700 and PE respectively.
Caution! Prepare (and process) compensation beads the same day of the flow cytometry assay.
16.1. To 100 uL of staining buffer, add both 1 drop of positive beads solution and 1drop of negative beads solution.
16.2. Add 2/10 uL of the corresponding antibody and homogenize.
16.3. Incubate 15 to 30 min at 4 ° C protected from light.
17) Add 500-600 mL of staining buffer to the microtubes obtained in step 15 and microtubes containing the beads.
18) Centrifuge for 5 min at 400 g 4 ºC and remove supernatant.
19) Add 1 mL of staining buffer and repeat step 18.
20B) Permeabilization if the permeabilization step is to be conducted at a later time:
20B.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.
20B.2 Centrifuge for 5 min at 400 g 4 ºC and remove supernatant.
20B.3 Add 500-600 µL of staining buffer and centrifuge for 5 min at 400 g 4 ºC
20B.4 Remove supernatant and repeat step 20B.3
20B. 5 Remove supernatant and resuspend cells in 500 µL staining buffer for storing cells at 4ºC for up to 72h.
Stop the protocol in this step for samples that do not need permeabilization and IL-10 staining.
20B.6 Centrifuge for 5 min at 400 g 4 ºC and remove supernatant.
20B.7 Add 250 uL of PermWash solution and incubate 15 min.
20B.8 Centrifuge for 5 min at 400 g 4 ºC and remove supernatant.
20B.9. Add 100 uL of PermWash solution plus 10 µL of anti-IL10-PE (or PE IC) antibody per million cells in the microtube.
20B.10. Incubate 30 min at 4 ° C.
20B.11 Centrifuge 5 min at 400 g at 4 ° C. Discard the supernatant and wash with 250 mL of PermWash solution.
20B.12. Discard the supernatant and wash again with 250 mL of PermWash.
20B.13. Discard the supernatant and resuspend in staining buffer.
OBSERVATIONS
In step 20B. 5 instead of stopping the protocol for blank, AF700 IC and CD19 samples, these were permeabilized by mistake. As a consequence, the gating strategy used in this assay must not be used in other experiments for non-permeabilized samples.
RESULTS
Voltages were set for both fluorocromes and their compensation was performed. As seen in the pictures above, both antibodies used in the assay work for dying CD19 and IL10 in PBMCs. In further analysis, the experiment will be repeated with isolated B cells (18/02/15).
