Materials and solutions: Live/dead kit L3224 Invitrogen: Calcein AM (4mM) and ethidium homodimer-1 (EthD-1) (2mM) PBS SFM Procedure: Incubate cells for appropriate time. Get the live/dead staining solutions to RT. Remove the media from your cells and washed with PBS 3X. Incubate the cells with PBS in the incubator for 5 minutes. To 3 ml of SFM add 1.5 l of Calcein AM (for a final concentration of 2 M) and 1.5 l of EthD-1 (for a final concentration of 1 M) and mix well. Note: These concentrations work well for AU565, BT549, HCC 1428, MDA-MB-231, BoM, HCC 70, HCC 1806, MDA-MB-468, BrM2a, HCC1395, LM2, SkBr3, BT474, HCC 1419, MCF7, ZR-75-1, SKVO-3, OVCAR-3, PC-3, and LNCaPcol. If your experiment does not work try a different range of concentrations (between 0.1 to 10 M for both Calcein AM and EthD-1). Remove PBS from cells and new solution of SFM. Incubate for 10 minutes. Take fluorescence imaging on a Zeiss Spinning Disc Cell Observer SD.
Materials and solutions: Live/dead kit L3224 Invitrogen: Calcein AM (4mM) and ethidium homodimer-1 (EthD-1) (2mM) PBS SFM Procedure: Incubate cells for appropriate time. Get the live/dead staining solutions to RT. Remove the media from your cells and washed with PBS 3X. Incubate the cells with PBS in the incubator for 5 minutes. To 3 ml of SFM add 1.5 l of Calcein AM (for a final concentration of 2 M) and 1.5 l of EthD-1 (for a final concentration of 1 M) and mix well. Note: These concentrations work well for AU565, BT549, HCC 1428, MDA-MB-231, BoM, HCC 70, HCC 1806, MDA-MB-468, BrM2a, HCC1395, LM2, SkBr3, BT474, HCC 1419, MCF7, ZR-75-1, SKVO-3, OVCAR-3, PC-3, and LNCaPcol. If your experiment does not work try a different range of concentrations (between 0.1 to 10 M for both Calcein AM and EthD-1). Remove PBS from cells and new solution of SFM. Incubate for 10 minutes. Take fluorescence imaging on a Zeiss Spinning Disc Cell Observer SD.
Cell_Staining_%28Ki67_%26_live-dead%29.docx