Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/02

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Summary

Following up on yesterday's transfection of U2OS cells, I'll be checking the transfection efficiency by observing the fluorescence of cells in culture via fluorescent microscopy. I'll also be advancing the KAH126/U2OS cell line, concluding the flow cytometry measurements of U2OS growth kinetics, and looking into making more KAH126 plasmid.

Goals

  • Advance KAH126/U2OS cell line from PS-α to PS-β (use 10cm TC-treated petri dish instead of T-75 flask)
  • Transfer transfected U2OS cells (and control) from 6 well plate to 10cm dishes
  • Photograph U2OS cells using phase & red fluorescence to assess transfection efficiency
  • Photograph SKNSH cells in 6 well plate to assess growth rate
  • Look into producing more KAH126 plasmid DNA
  • FC of 3 U2OS wells from 24-well plate

Protocol

advancing adherent cells
detecting fluorescent protein expression
transformation of E. coli with plasmid DNA
To advance the transfected U2OS cells from 6-well plates to 10cm petri dishes:

  1. Warm up: PBS, trypsin buffer, U2OS medium (10%FBS, 1%p/s)
  2. Harvest DNA-positive cells from 6-well plates: aspirate off medium, wash with 2 mL PBS, trypsinize with 0.5 mL trypsin/EDTA
  3. Label 3 10cm round TC-treated sterile petri dishes. Transfer 6 mL medium into each.
  4. Collect trypsinized cells from each small well: use 3.5 mL medium, pipette up and down to make an even cell suspension. Transfer into the 6mL medium in one of the 10 cm plates. Spread cells by moving plate up/down/back/forth. Repeat for two remaining samples.
  5. Put plates in incubator. Check once a day for RFP.

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

Fluorescence microscopy images shown below. Wells 3 and 4 appear to have high transfection efficiencies, well 5 less so. Negative control has no fluorescence (not shown).

Well 3

Well 4

Well 5


Flow cytometry data for the last day of U2OS growth kinetics:

Plot 1 (FSC-A/SSC-A)
All P2
Count Volume (µL) Events / µL Count Events / µL % of All
A01 Blank 736 100 7 0 0 0.00%
A02 Well A3 25,846 100 258 20,249 202 78.34%
A03 Well B3 23,578 100 236 19,179 192 81.34%
A04 Well D3 21,960 100 220 18,169 182 82.74%



Average cell density is 1.92E5+/- 1E4 cells/mL. In a 6-well plate this would translate to >7.5E5 cells per well.

Here's the results of 5 days of sampling the 24-well plate for measuring U2OS growth kinetics:


Entering this data into an online doubling time calculator gives a doubling time of 17.51 hours (Roth V. 2006 <http://www.doubling-time.com/compute.php>).

And for future reference, here's a sheet of all the data from the past 5 used to generate the above graph. Note that samples for each day were taken in triplicate from randomly-chosen wells in the same 24-well plate, with the exception of Day 0, which has only one data point (measurement of the parent culture right before seeding the plate).

U2OS Growth Curve Concentration (cells/mL)
Date Day Well 1 Well 2 Well 3 Average St Dev
12/29/14 0 5.0E+03 5.0E+03 #DIV/0!
12/30/14 1 7.0E+03 1.7E+04 1.6E+04 1.3E+04 5.5E+03
12/31/14 2 2.6E+04 2.7E+04 1.4E+04 2.2E+04 7.2E+03
1/1/15 3 7.4E+04 7.7E+04 6.8E+04 7.3E+04 4.6E+03
1/2/15 4 2.0E+05 1.9E+05 1.8E+05 1.9E+05 1.0E+04


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