Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/30

RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines

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Summary Checking on the U2OS & SKNSH cell lines today. I'll take three wells from the 24-well plate and 1 well from the 6-well plate, trypsinize the cells to get them into solution, then measure their density on the flow cytometer. Should be able to get a rough idea of when the plates will be ready for transfection then. I'll also be taking pictures of the plates in order to correlate measured cell density (via FC) with estimated confluence. Goals Use flow cytometry to measure cell density of U2OS cultures in wells Take photographs of U2OS & SKNSH cells in wells Protocol flow cytometry for mammalian cells Materials List materials here, to be gathered before the experiment Consumables such as media, antibiotics, DNA, kits etc. Notes Observations, measurements, placeholder info etc. go here Results/Conclusions Photographs of SKNSH & U2OS cultures on the right. SKNSH culture is actually at much lower confluency than depicted, with large regions of empty space in the well. SKNSH U2OS Flow cytometry data, from one well from a 6-well plate and three wells from a 24-well plate. P2 is a gate set up to capture all mammalian cells in the sample. Plot 1 (FSC-A/SSC-A) All P2 Count Volume (µL) Events / µL Count Events / µL % of All A01 Blank 661 100 7 0 0 0.00% A02 Well 1 (on 6-well plate) 7,757 100 78 5,967 60 76.92% A03 Well B4 (24-well plate) 1,609 100 16 654 7 40.65% A04 Well C5 (24-well plate) 2,590 100 26 1,694 17 65.41% A05 Well D6 (24-well plate) 3,159 100 32 1,595 16 50.49% The 6-well plate contains 4mL of medium per well, whereas the 24-well plate has 1mL of medium per well. Therefore to determine the concentration of cells/mL, divide the 6-well plate number by 4. This means that the concentration of cells in the 6-well plate is 15,000 cells/mL (60,000 cells total) and in the 24-well plates is an average of 13,300 cells/mL (stdev 5.5 cells/mL). Since we're looking for an ideal cell concentration of 125-250,000 cells per well in a 6-well plate, the U2OS culture should be ready for transfection tomorrow or Thursday at the latest. Will repeat the flow cytometry / microscopy tomorrow and make a decision then.