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5-Ethynyl-2′-deoxyuridine (EDU) proliferation assay

AM Alaknanda Mishra
KM K. Varsha Mohan
PN Perumal Nagarajan
SI Srikanth Iyer
AK Ashwani Kesarwani
MN Madhu Nath
LM Laxmi Moksha
JB Jashdeep Bhattacharjee
BD Barun Das
KJ Kshama Jain
PS Parul Sahu
PS Prakriti Sinha
TV T. Velapandian
PU Pramod Upadhyay
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EDU (5-ethynyl-2′-deoxyuridine) cell proliferation assay was performed using Click-It EDU-Alexa Fluor 488 assay kit as per the manufacturer’s instruction (Thermo fisher scientific, USA). Briefly, the cells were plated at an optimum density and EDU was added to the culture medium at a concentration of 10 μM, mixed well and incubated for 1–2 h. The EDU-treated cells were washed with 3 ml of 1% BSA in PBS and cells were pelleted. One hundred microlitres of fixative (Component D) was added to the pellet, vortexed and incubated for 15 min at RT in dark followed by a wash with 1% BSA in PBS and centrifuged to obtain a pellet. The cell pellet was resuspended in 1X Click-It permeabilization buffer and incubated for 15 min at RT. The permeabilized cells were washed with 3 ml of 1X Click IT saponin-based wash reagent, centrifuged and resuspended in PBS. The samples were run in FACS Verse and analysed by Flowjo 10 software for Alexa Fluor 488 positive cells.

Copyright and License information: The Author(s) ©2020
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