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Contents |
Microscopy
Protocol: Microscopy and slides
Preparation of slides
- Clean glass slides with 95% ethanol and leave it under the hood for air drying
- Label the slides with the sample name and date</pre>
Methanol Fixing of Cells
- Add 500 µl of PBS in 1.5 ml eppendorf tubes.
- Take required volume from cell culture to obtain 1000, 500 and 250 cells and add it to the PBS
- Centrifuge at 450 rcf for 5 min
- Remove excess PBS manually with a pipet. Make sure you do not touch the cell pellet (may be visible as a tiny dot) (PBS wash 1)
- Re-suspend the pellet in 500 µl of PBS.
- Centrifuge at 450 rcf for 5 min
- Remove excess PBS manually with a pipet. Make sure you do not touch the cell pellet (may be visible as a tiny dot) (PBS wash 2)
- Add 20 µl of ice-cold methanol to the cells and re-suspend. Let it rest at -20°C for 10 min
- Centrifuge at 450 rcf for 5 min
- Remove excess methanol manually with a pipet.
- Re-suspend the pellet in 500 µl of PBS.
- Centrifuge at 450 rcf for 5 min
- Remove excess PBS manually with a pipet.
- Re-suspend the pellet in 500 µl of PBS.
- Centrifuge at 450 rcf for 5 min
- Remove excess PBS manually with a pipet.
- Re-suspend the pellet in 10 µl of PBS.
- Drop the pellet with PBS on the glass slide
- Cover it with cover slip.
- Store it at 4°C.
Now the slides can be taken to the BIOP facility at EPFL and imaged with a confocal microscope.
The slides that had been made made on the previous day (with methanol and formaldehyde) have been taken to the BIOP facility.
Passaging
Protocol: Cell Passaging
These are the general mammalian cell passaging rules, they work for CHO cells and HEK cells.
The cells should be left to grow for two days, after what their density should be reduced and the medium should be changed.
We usually bring them either to 2 mio/ml, either to 1 mio/ml.
- Put the volume of medium that corresponds to the amount of cells you'd like to passage at 37°C, leave it there to heat for 10 minutes (more for bigger volumes, less for smaller).
- Measure the PCV of your current cell sample. Usually, the cells double every day, so you can estimate their number, but a PCV is more precise. Take 200 µl of cell suspension in a mini-PCV tube, centrifuge them (1 min at 5000 rpm), the cells will fall down in the thin tube at the bottom. Use the VoluPAC Reader to measure the PCV (the volume in µl the centrifuged cells take).
- If you're dealing with HEK cells, the PCV is also the number of millions of cells/ml you have.
- If you have CHO cells, divide the PCV by 0.4 to obtain the mio/ml cell density.
- Take the volume that will contain the amount of cells you need from the original sample in a centrifuge tube, centrifuge it at 1500 rpm for 3 minutes. A pellet will form.
- Remove the supernatant (under the hood) either with the vacuum pump, either by simply discarding it.
- Resuspend in 10 ml of warmed medium (pipet up and down)
- Add the remaining medium, mix again
- Split the cultures into 10 ml samples in separate tubes, put them in the shaker at 37°C, passage again after 2 days.
CHO and HEK were passaged.
