Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells

Protocol tips

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	Supplement 6 ml PBS with 6 μl Ethidium homodimer III (EthD-III) and 3 μl Calcein AM. Incubate the cells for 30-45 minutes at room temperature

Protocol tips
Supplement 6 ml PBS with 6 μl Ethidium homodimer III (EthD-III) and 3 μl Calcein AM. Incubate the cells for 30-45 minutes at room temperature

Publication protocol

The cytotoxicity of the AMPDs was tested on the human progenitor skin cells (passage 8) cultured directly on the collagen membranes. Briefly, biological bandages were prepared as previously described, then for each AMPD 2 mg were dissolved in 10 ml culture medium in order to obtain a concentration of 200 μg/ml. Two-fold dilutions were performed to obtain AMPDs solutions of 100 and 50 μg/ml. AMPDs solutions were added to bandages and were incubated at 37 °C for 24 hours. Then, cell viability was assessed using the Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (Biotium, Hayward, CA, USA). Reagent solutions were prepared according to manufacturer protocol, where 6 ml PBS was supplemented with 6 μl Ethidium homodimer III (EthD-III) and 3 μl Calcein AM. After washing with PBS and 45 minutes incubation of the bandages in reagent solutions, images were acquired with the microscope (Zeiss Axiovert 100, Oberkochen, Germany). For the negative control, methanol was added 30 minutes after the washing step with PBS.

Cell viability was also tested on adult fibroblasts (NP/JATH) seeded in 48-well plates at a cell density of 6,500 cells/cm2. AMPDs solutions were prepared as previously described for the bandages to obtain concentrations of 100 and 50 μg/ml (n = 6 wells/condition), which were added to cells 24 hours after seeding. Cell viability was measured after 24 hours using the CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). Reagent solution was prepared according to manufacturer’s instructions. 100 μl were then distributed to each well of the plate and incubated for 1 h30. 90 μl of supernatant was placed in a new 96-well plate and the absorbance at 490 nm was measured with a spectrophotometer (Wallac Victor2 1420 multilabel counter, PerkinElmer, MA, USA). For the negative control, 100 μl methanol was added 30 minutes before measurement after discarding the culture medium. Before adding the reagents used for the measurements, the culture medium of each well was aspirated and discarded and the wells were washed with 100 μl PBS (GIBCO® PBS, pH 7.4 (1×), Life Technologies, Carlsbad, CA, USA).

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