Question

How much time required to grow mdck cell in cell culture?

Answer 1

This chapter explains how the Madin-Darby canine kidney (MDCK) cell is grown to examine epithelial cell biology. Epithelial cells display a structural and functional polar organization. Two different strains of the MDCK cell are known. Strain I cells are derived from a low-passage MDCK cell stock. Strain II cells form a monolayer of lower resistance. MDCK strain II cells have been used primarily for studies of the cell biology of epithelial cells. Transcytosis is, however, studied more conveniently in MDCK strain I cells because of their high electrical resistance. MDCK Cells need to be seeded on polycarbonate filters at high density, higher than that achieved by confluent cells on plastic. The cells form tight junctions within 24 h and reach the maximum tightness on the filters in 4 days

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Answer 2

MDCK cells in 12-well plate and make sure next day wells are a confluent monolayer. Then I aspirate the cell culture supernatant and wash with PBS twice, and add 200 ul of tenfold virus dilutions to each well and incubate for an hour in 37 at RT for 1h and rock the plate side to side every 10 min. Then I mix and add the agar overlay which consists of sterile water, 2x MEM, 1% DEAE dextran, 5% sodium bicarbonate, 1 ug/ml trypsin-TPCK, and 2% Oxoid agar. The agar solidifies at room temperature for 15 minutes before placing in the incubator. After 24 hours, the cells are all dead. So I have tried a lot conditions (to make sure the agar was not too hot and to change ingredients of agar overlay). Finally, I found all MDCK cells without trypsin-TPCK in agar overlay survived. Does someone can tell me what happened to my trypsin-TPCK (they were prepared in 2016 and stored in -20). And Now I have ordered new trypsin-TPCK, the instruction showed it should be dissolved in 1mM HCl (ph=3).