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Contents |
Morning: Cell counting and sampling
- Cell counting
Given our cell density we used a 12x dilution (table). We transfered 6 samples of cells into a 96 well plate, added Trypan blue and analysed each sample with Countess.
| Sample # | Viable cells | Dead cells | Viability | Total cells |
| 1 | 182 | 12 | 94% | 194 |
| 3 | 112 | 7 | 94% | 119 |
| 5 | 124 | 8 | 94% | 132 |
| 7 | 129 | 16 | 89% | 145 |
| 9 | 170 | 3 | 98% | 173 |
| 11 | 127 | 9 | 93% | 136 |
- Average cell # for samples 3.5.7 = 122 cells
- LCD: 122*12/400 = 3.66 mio cells/ml
-> we need to take 1.37ml to obtain 5 mio cells.
- Comments
Sample 7 has been excluded because of bubble-like structures which appeared while scanning and were confused with cells clusters by the program.
- Western Blot sampling
Protocol (add to separate page):
- Centrifugation of the cell samples at 2500xg for 10min. Discard the supernatant (vacuum).
- Washing: Resuspend the pellet in PBS1x, centrifuge at 2500xg for 10min, discard supernatant.
- Add at least 1mL PER-M Reagent for 100mg (~100µl) of cell pellet. Pipette up and down to suspend.
- Shake gently for 10min.
- Remove cell bedris by centrifugation: ~1400xg for 15min
- Transfer the supernatant into new tubes for analysis or storage.
- Samples where taken from odd numbered tubes : 1,3,5,7.
- We used 300µl of M-PER Reagent based on the size of our pellets.
- Maximum centrifugation was 10'600xg.
Protocol: None
Forgot to insert protocol.
- Comments
For samples 3 and 5 we had to repeat step 5 (centrifugation to remove cell debris) because the cell pellet got aspired in the pipette while trying to transfer the supernantant into fresh tubes.
Afternoon: Cell lysates
We prepared cell lysates for future mRNA sampling and for analysing fluorescence the same day.
- Protocol: Sampling for Tecan machine (add on separate page)
- Take ~1mio cells in 750 µl PBS
- Centriguge samples at 450xg for 5min
- Remove the supernatant (vaccum)
- Resuspend in 200µl of 1% TritonX in PBS
- Load onto black well and leave on the shaker for 1 hour.
- Read fluorescence on Tecan machine
- Results from the Tecan machine: File:Team-EPF.Lausanne-iGEM 19.07.12 LOVTAP, RFP.xls
Protocol: Sampling for mRNA (add on separate page)
- Take ~1mio cells in 750 µl PBS
- Centriguge samples at 450xg for 5min
- Remove the supernatant (vaccum) leaving 50µl.
- Vortex and resuspend.
- Add 1µl PBS, vortex again.
- Centrifuge at 450xg for 5min
- Remove supernatant (pipette) and freeze in liquid nitrogen. Store at -20°C.
Protocol: None
Forgot to insert protocol.
- Comments
Our cell density was 6mio cells/ml, therefore we needed to take a volume of 170µl to have 1mio cells/ml.
