Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/05/29

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Overview

Today I'll be checking the transfection efficiency of the neon transfection system on K562 cells using fluorescence microscopy and flow cytometry, then moving forward from there depending on the results.

Fluorescence Microscopy

  • pMax GFP, 1000 v, 50 ms, 1 pulse

2015-05-29 K562 Well 1 GFP phase 10x.jpg

  • pMax GFP, 1350 v, 10 ms, 4 pulses

2015-05-29 K562 Well 3 GFP phase 10x.jpg

  • pMax GFP, 1450 v, 10 ms, 3 pulses

2015-05-29 K562 Well 5 GFP phase 10x.jpg

  • KAH126-MV2, 1000 v, 50 ms, 1 pulse

2015-05-29 K562 Well 7 mCherry phase 10x.jpg

  • KAH126-MV2, 1350 v, 10 ms, 4 pulses

2015-05-29 K562 Well 9 mCherry phase 10x.jpg

  • KAH126-MV2, 1450 v, 10 ms, 3 pulses

2015-05-29 K562 Well 11 mCherry phase 10x.jpg

Note: Even numbered wells were replicates of the preceding wells. None of them showed significant transfection efficiencies. This is due to pipetting error during the neon transfection protocol - there wasn't enough of the DNA/cell mixture to completely fill the pipette tip for the duplicates. Next time fix this by incorporating enough of a buffer for there to be some cells/DNA left over after all replicates.

Flow Cytometry

Plot 1 (FSC-A/SSC-A) Plot 3 (FL1-A [GRN]) Plot 4 (FL3-A [RED])
All P2 M2 M1
Sample ID Count Volume (µL) Events / µL Count Events / µL % of All Count % of This Plot Count % of This Plot
1. pMax GFP, 1000 v, 50 ms, 1 pulse 1,841 10 184 1,129 113 61.33% 792 70.15% 365 32.33%
2. pMax GFP, 1350 v, 10 ms, 4 pulses 1,240 10 124 736 74 59.35% 696 94.57% 553 75.14%
3. pMax GFP, 1450 v, 10 ms, 3 pulses 520 10 52 60 6 11.54% 54 90.00% 48 80.00%
4. KAH126-MV2, 1000 v, 50 ms, 1 pulse 774 10 77 200 20 25.84% 3 1.50% 13 6.50%
5. KAH126-MV2, 1350 v, 10 ms, 4 pulses 1,035 10 104 112 11 10.82% 3 2.68% 13 11.61%
6. KAH126-MV2, 1450 v, 10 ms, 3 pulses 1,002 10 100 56 6 5.59% 1 1.79% 12 21.43%
Untreated 21,387 10 2139 15,909 1591 74.39% 8 0.05% 24 0.15%

Counts are low due to low cell density and volume (only 50 µL of each sample was used to measure transfection efficiency). Despite that, initial results look promising. Methods 2 and 3 both have high transfection efficiencies for pMaxGFP (95% and 90%, respectively) and KAH126-MV2 (12% and 21%, respectively). Will have to repeat using higher concentration KAH126-MV2 plasmid; 809 ng/µL is a little on the low side.

There's also a lot more debris / dead cells in the cultures treated with methods 2 and 3. Need to followup with a cell viability assay in the future as well.