Multi-parametric staining of cryopreserved U937 cells. U937 cells were loaded into 100 μm i3C and underwent a freezing - thawing cycle. After 24 hours of post-thawing culture, the U937 cells were stained sequentially first by TMRM and Annexin V and next by FDA and PI. Transmitted light (A) and fluorescent images of U937 cells stained with TMRM and Annexin V (B), and FDA (C) are presented. Arrows indicate different cell statuses: Blue - high functional activity - morphologically intact, highly TMRM and FDA positive, Annexin V negative; Yellow - early apoptosis - a morphologically changed cell with a slightly condensed nucleus, exhibiting low TMRM staining, FDA positive staining and strong Annexin V staining; White - late apoptosis - a morphologically changed cell with nucleus defragmentation and pronounced Annexin V positive staining, TMRM and FDA negative staining. Scale bar: 0.05 mm. (D) Scatter diagram of individual TMRM (FI) values vs. esterase enzymatic activity values (slopes), measured in post-thawed U937 cells 24 hours after cell thawing. Note the detection of 4 cell subgroups based on their functional activity parameters.
Afrimzon et al. BMC Cell Biology 2010 11:83 doi:10.1186/1471-2121-11-83