Apoptosis and Necrosis Quantification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
• Kit provides a convenient assay for quantifying apoptotic (green) and necrotic (red) cells within the same cell population by flow cytometry or fluorescence microscopy.	• Seed 1 × 10^3 MiaCaPa-2 cells in each well.

Upstream tips
• Kit provides a convenient assay for quantifying apoptotic (green) and necrotic (red) cells within the same cell population by flow cytometry or fluorescence microscopy.
Protocol tips
• Seed 1 × 10^3 MiaCaPa-2 cells in each well.

Publication protocol

In a 96 well-plate, 200 μL of fresh medium (i.e. DMEM containing 10% FBS, 1% glutamine, 1% penicillin) containing 1 × 10^3 MiaCaPa-2 cells was plated in each well and incubated for 24 h at 37 °C in a 5% CO2 atmosphere. Then, 200 μL of the medium of each well was replaced with 200 μL of freshly prepared solution containing GEM·HCl, GEM–monomer conjugate NPs, GNPs and hybrid NPs of Sample S7′ at their IC50. The cells were further incubated for 72 h; subsequently, the total amount of media was removed and cells were washed twice with PBS. The staining solution (i.e. 5 μL of Annexin V solution, 5 μL of Ethidium Homodimer solution and 100 μL 1× binding buffer) was added and the cells were incubated for 15 min protected from light. The staining solution was removed and cells were washed with 1× binding buffer. The cell fluorescence was recorded using EVOS FL and ImageJ software was used to quantify the necrosis and apoptosis. Furthermore, microscopic images of the cells that were incubated with the hybrid NPs and GNPs were recorded and imageJ software was used to quantify the GNP binding/uptake per cell. More precisely, the GNPs were thresholding in red and the total red signal area was measured in pixel units.


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