OBJECTIVE

To set-up a sorting protocol to isolate the B-cell subset responsible of producing pathogenic antibodies in neuropathies.

The whole process is copied/adapted from Chihara et al paper on NMO (see reference)

MATERIALS

• 10 Heparin-lithum tubes with 10mL blood each to isolate PBMCs
• CD19-FITC, CD27-PerCPCy5.5, CD38-PE (all from BD)
• recombinant human IL-6 (Preprotech)
• Basal medium (RPMI + 10%FBS + 2mM L-Glutamine + P/S)

PROCEDURE

•  Draw blood the day before an let O/N rocking.
• Transfer all blood to 2 50mL falcons and spin down cells at 2000g 10′
• Remove plasma and store it
• Add RPMI (w/o additives) up to 50mL again and re-suspend.
• Prepare 4 50mL Falcons with 10mL Lymphoprep each.
• Add 25mL of cell suspension to each Falcon w/ Ficoll and spin at 400g 20′ w/o brake
• Get PBMCs and re-suspend in RPMI. Spin down at 300g to wash (2x)
• Re-suspend in 5mL and count (counted 45xmill cells)
• Prepare these tubes

Tube 1: CD19-FITC only

Tube 2: CD27-PerCPCy5.5 only

Tube 3: CD38-PE only

Tube 4: Only cells

Tube 5: All antibodies

• All compensation tubes 1mill cells (in 1mL)
• The rest of cells (45mill aprox.) into 3mL of RPMI for sorting
• Added 5uL of each antibody for compensation and 180uL for the sorting tube
• Incubate Abs 30min in the dark
• Wash 2x with RPMI
• Prepare 3 tubes (CD19+CD27+CD38+, CD19+CD27+CD38-, CD19+CD27-CD38-) with basal medium (plus 1ng/mL of IL6)
• Go to sort
• After sorting spin down cells at 400g and re-suspend in 100uL of basal medium plus IL6 (1ngr/ml) and count
• Plate 100uL with 50.000 cells from each subset in a 96-well plate with basal medium + IL6. Wait one week to collect supernatant.

RESULTS

Starting with 45mill cells i was able to obtain 35000 from the CD19+CD27+CD38+ population, 400.000 from the CD19+CD27+CD38- and 500.000 from the CD19+CD27-CD38-.

Attachement 1

Attachement 2

CONCLUSIONS

It looks that with approximately 100mill cells we can be able to have no more than 50.000 plasmablasts from a HD.