B/T lymphopoiesis potential assay protocol

Culture base media: α-MEM + 20% FBS + 1% Pen/Strep

B cell assay

  • OP9
  • Base media + 10ng/ml Flt3L + 10ng/ml IL-7
Day -1:
  • Seed 12.5K cells per well of 96-well flat-bottomed plate in 150µl base media
    • Preferably 100% confluence so that sorted cells do not differentiation on plate
Day 0:
  • Sort cells (1 HSC) into each well directly
  • Add 100µl media per well with 2.5x cytokines
Maintenance:
  • Change media every 4 days by gently removing 100µl and adding 100µl media with 1x cytokines
Analysis: 12-15 days for HSCs
  • Lay out FACS tubes in a 96-well configuration and cover all tubes with a piece of 100µm filter
  • Pipette samples through filter with an automatic pipette
  • Analyze by flow cytometry
    • CD45.2: AF700
    • CD19: APC
    • B220: FITC
    • DAPI

T cell assay

  • OP9-delta
  • Base media + 5ng/ml Flt3L + 5ng/ml IL-7
Day -1:
  • Seed 12.5K cells per well of 96-well flat-bottomed plate in 150µl base media
    • Preferably 100% confluence so that sorted cells do not differentiation on plate
Day 0:
  • Sort cells (1 HSC) into each well directly
  • Add 100µl media per well with 2.5x cytokines
Maintenance:
  • Change media every 4 days by gently removing 100µl and adding 100µl media with 1x cytokines
Analysis: 28 days for HSCs
  • Lay out FACS tubes in a 96-well configuration and cover all tubes with a piece of 100µm filter
  • Pipette samples through filter with an automatic pipette
  • Analyze by flow cytometry
    • CD45.2: AF700
    • CD4: APC
    • CD8a: FITC
    • DAPI