Shreffler:BCell Staining

From OpenWetWare

Jump to: navigation, search

Contents

Overview

Staining B lymphocytes, gating for plasmablast population

Antibody Panel

  • CD3-FITC (BD: 555332), 10 µl/test
  • CD14-FITC (BD: 555397), 10 µl/test
  • CD19-APC-Cy7 (BD: 557791), 2.5 µl/test
  • CD27-PE (BD: 555441), 10 µl/test
  • CD38-APC (BD: 555462), 10 µl/test
  • IgM-PE-Cy5 (BD: 551079), 10 µl/test

Explanation

CD3 and CD14 (both on FITC) function as a dump gate. Assuming cells are taken from whole blood, CD19+ cells are B cells. Of these, CD27+ cells are memory B cells, containing the CD38-high plasmablast sub-population. CD27- cells consist of naive B cells. Finally, plasmablasts should be IgM negative, but at the moment we aren't able to acquire neat IgM+/IgM- bimodality of CD27+ cells unless the stained cells are CD19+ cells selected using StemCELL enrichment kit.

Staining Protocol

Pipette 200 µl of whole blood into 10 PS tubes. Of these, four will be stained with the entire panel (run these as the same sample, to acquire ~1000 plasmablasts). Five tubes are FMOs, one for each fluorescent label. The last tube is an unstained control. Flow up to 24 hours later, though longer is still viable.

Personal tools