Calcein AM Cell Viability Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 100 uL 2uM Calcein AM in PBS to each well. Incubate at 37°C for 1h .	Serum in cell culture medium may contain esterase activity, which can increase background fluorescence. Cells can be rinsed in PBS at this step to reduce background caused by residual serum

Protocol tips
Add 100 uL 2uM Calcein AM in PBS to each well. Incubate at 37°C for 1h .
Downstream tips
Serum in cell culture medium may contain esterase activity, which can increase background fluorescence. Cells can be rinsed in PBS at this step to reduce background caused by residual serum

Publication protocol

Murine 3T3-L1 fibroblasts (American Type and Culture Collection) were induced to differentiate as previously described;5 adipocyte CM from these cells was collected, centrifuged and used for most assays. Fibroblast-CM was used as a control. Murine mammary cancer cells (M-Wnt, E-Wnt) and human breast cancer cells (MCF-7) were grown in RPMI 1640 and DMEM media, respectively, at 37°C with 5% CO2. The media were supplemented with 10% FBS and 1% penicillin/streptomycin. Cell proliferation was assessed using a Calcein AM cell viability assay kit (Biotium) where the fluorescent signal is monitored using 485 nm excitation wavelength and 530 nm emission wavelength according to manufacturer’s protocol. Briefly, cells were seeded at a density of 3 × 103 cells/well on 96-well flat bottom plates (Falcon, Becton Dickinson Labware) and allowed to adhere for 24 h. Cells were cultured with 10% FBS, 0.1% adipocyte-CM or 0.1% fibroblast-CM. After 48 h of continuous culture, 100 μl of Cacein AM in PBS (2 μM) was added to each well and incubated for 1 h at 37°C. Fluorescence intensity was determined using a BioTek Synergy Multi-Mode Microplate Reader.

In order to evaluate the effect of adipocyte-CM on the cytotoxicity of gemcitabine HCl or GemC18-NPs, cells (M-Wnt, E-Wnt or MCF-7) were cultured in the presence of adipocyte-CM or fibroblast-CM (1%) and gemcitabine HCl or GemC18-NPs for 48 h (the molar concentration of gemcitabine was 50 μM). Cell proliferation was measured using the Cacein AM cell viability kit as above.

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