avoid extended exposure to light. Dye stocks can be either
wrapped using aluminum foil or stored in amber-colored tubes.- Degassing of DMSO and autoclaved water used for preparing
the stock solutions for CuAAC reaction is recommended.
However, in our cell-based experiments we have observed
efficient staining by click reaction even when reagents stocks
were not degassed. - DAPI is light sensitive therefore all stock solution preparation
and staining should be performed in the dark. Store the stock
solutions at 4C in amber-colored plastic vials. - Freshly prepare stock solutions in dark and inside cell culture
hood. Working solution to stain DNA in live cells must be
thawed in a 37C water bath. Make sure that the staining
solution is warm (~37C) to avoid heat shock to cells. Store
the stock solutions at 20 C in amber-colored plastic vials. - Typically we use 100 mm dish for culturing the cells. Depend-
ing on the cell count prepare cell suspension in the complete
media required for seeding into 6-well dish. Prior to seeding,
place sterile glass coverslip in each well and pipette the media
containing the cells at the center of the well. Swirl the plate very
carefully to make sure uniform distribution of cells on glass
coverslip. Place the plate in the incubator after properly label-
ing the wells. The incubation time should be optimized to get
~70% cell confluence. - After washing the cells with 1PBS, the fixation, permeabili-
zation, and click staining can be performed outside the cell
culture hood. - Always perform a control staining reaction with cells treated
with transfecting mix without AMUTP. - The incubation time should be optimized to get ~70% cell
confluence. - It is best to image live cells after click and Hoechst staining
immediately to minimize cell death.
Acknowledgments
This work was supported by the Department of Science and Tech-
nology, India, SERB grant (EMR/2014/000419) to S.G.S. and
the Centre of Excellence in Epigenetics grant from the Department
of Biotechnology, Government of India to S.G. The authors wish
to thank Arun Tanpure, Progya Mukherjee, Soumitra Athavale,
Rahul Jangid, and Ashwin Kelkar for discussion and help with
work that has led to the optimization of these protocols.370 Anupam A. Sawant et al.