HL-60 differentiation to neutrophils/monocytesΒΆ

Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom

Differentiation of HL-60 to neutrophil or monocyte-like cells by Vitamin A (ATRA) and Vitamin D (1a,25(OH)2D3)

  • Seed cells at 2.5x105 cells/ml as 4ml cultures in 25cm2 flasks.

If seeding to plates, seed to base-density of approximately 1x106 cells/25cm2 e.g. 1cm2 well = 3.14cm2 25/3.14 = 8 1/8 = 0.125 x 10^6 cells/well

  • Differentiation can be induced with 100nM ATRA (neutrophils) or 100nM 1a,25(OH)2D3 (monocytes)
  • Re-feed cells late on day 2 or morning day 3, ensuring stocks stay below 1x106/ml to avoid spontaneous differentiation
  • First signs of differentiation will appear day 3
  • Complete differentiation should be apparent day 5