I recently tried a protocol to differentiate HL60 cells to neutrophil-like cells using 1uM ATRA. I have 2 questions:
- How long do you keep cells with ATRA? I first fed the cells with ATRA and later on day 2, I refed them again with ATRA-supplemented media. Wondering if just 1 dose of ATRA is enough? and I can remove the media with ATRA on day 2/3 and refeed cells with regular media?
- Given the above protocol, I noticed significant cell death at day 6 (about 40%) and all cells died at day 8. Cells were still ok at day 5. Density was maintained at less than 500,000 cells/ml throughout. Differentiation was confirmed with Giemsa staining and Cd11b flow. So I just wonder why my cells died so quickly after differentiation and is there a way to boost viability?
I refer, exactly, from an article:
" Differentiate cells in culture medium containing 1.3% DMSO. Because DMSO is more viscous and denser than culture medium, premix medium with DMSO before adding cells. Cells stop proliferating upon differentiation and typically achieve a density of 1–2 million cells/ml at 7 days postdifferentiation (Fig. 1). Cells are most active 5–6 days postdifferentiation, but can still respond even after 8 days (see Note 9). "
The article can be found here:
Article Chemotaxis in Neutrophil-Like HL-60 Cells
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