Cell Viability

viability

1. Hepatic stellate cell viability
Trypan blue assay
Cell viability is measured following the trypan blue protocol. Activated HSCs are seeded in 12 well plates and incubated for 4 hr (standardization for individual drug) with drug. Next, the cells are treated with trypan blue (0.04 mg/ml) and incubated for another 15 min. Bright field images are taken using Olympus inverted microscope. The number of cells having a blue nucleus is counted and percentage of dead cells is being plotted in the form of a graph (Strober, 2001).
CMFDA based assay
Cell viability is measured using 5- chloromethylfluorescein diacetate probe (CMFDA). Here, activated HSCs are subjected to drug treatment for 4 hr (standardization for individual drug), followed by incubation with 5 μM CMFDA for 2 hr. Next, the cells are washed with PBS and images are taken using DP71 camera adapted to an Olympus IX71 microscope (Johnson et al 2013).
2. Measurement of Apoptosis
(i) PI Incorporation Assay
Activated HSC cells are treated with drug for 4 hr (standardization for individual drug), incubated with 1 μM of propidium iodide (PI) for 10 mins. To remove unbound PI, cells are washed with PBS gently and the fluorescent images are acquired with DP71 camera fitted with an Olympus IX71 microscope. The number of PI positive cells are determined for 5 different fields of 5 independent experiments and a graph is plotted. (Majumder et al 2011).
(ii) Annexin V-FITC Apoptosis Detection Kit
Activated HSCs are treated with drug for 4 hr (standardization for individual drug) and are being processed according to the protocol supplied by the manufacturer (Calbiochem, Germany). Fluorescence images of the cells are captured and the number of annexin V-FITC-positive cells are counted per field.

REFERENCES
1. Strober W. Trypan blue exclusion test of cell viability.Curr Protoc Immunol. 2001 May;Appendix 3:Appendix 3B.
2. Johnson S, Nguyen V, Coder D.Assessment of cell viability.Curr Protoc Cytom. 2013;Chapter 9:Unit9.2.
3. Majumder S, Siamwala JH, Srinivasan S, Sinha S, Sridhara SR, Soundararajan G, Seerapu HR, Chatterjee S. Simulated microgravity promoted differentiation of bipotential murine oval liver stem cells by modulating BMP4/Notch1 signaling. J Cell Biochem. 2011 Jul;112(7):1898-908.