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 analyzed each sample with Countess.

• 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:

1. Centrifugation of the cell samples at 2500xg for 10min. Discard the supernatant (vacuum).
2. Washing: Resuspend the pellet in PBS1x, centrifuge at 2500xg for 10min, discard supernatant.
3. Add at least 1mL PER-M Reagent for 100mg (~100µl) of cell pellet. Pipette up and down to suspend.
4. Shake gently for 10min.
5. Remove cell debris by centrifugation: ~1400xg for 15min
6. Transfer the supernatant into new tubes for analysis or storage.

• Samples were 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.

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.

• The results from the Tecan plate reader did not show any significant increase in red fluorescence for co-transfected cells. We were also not able to detect intrinsic fluorescence in the range of the Lov domain.

Protocol: Sampling for mRNA

1. Take ~1mio cells in 750 µl PBS
2. Centriguge samples at 450xg for 5min
3. Remove the supernatant (vaccum) leaving 50µl.
4. Vortex and resuspend.
5. Add 1µl PBS, vortex again.
6. Centrifuge at 450xg for 5min
7. Remove supernatant (pipette) and freeze in liquid nitrogen. Store at -20°C.

Passaging

Our cell density was 6 mio cells/ml, therefore we needed to take a volume of 170 µl to have 1 mio cells/ml.