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

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RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines Main project page
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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
SKNSH

U2OS
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)
AllP2
CountVolume (µL)Events / µLCountEvents / µL% of All
A01 Blank6611007000.00%
A02 Well 1 (on 6-well plate)7,757100785,9676076.92%
A03 Well B4 (24-well plate)1,60910016654740.65%
A04 Well C5 (24-well plate)2,590100261,6941765.41%
A05 Well D6 (24-well plate)3,159100321,5951650.49%

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.



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