Preparation of live cell samples for uorescence spectroscopy and computational super-resolution imaging

Jagadish Sankaran National University of Singapore https://orcid.org/0000-0001-7021-9807 Harikrushnan Balasubramanian National University of Singapore https://orcid.org/0000-0003-4966-0221 Wai Hoh Tang National University of Singapore https://orcid.org/0000-0002-6717-9426 Xue Wen Ng National University of Singapore Adrian Röllin National University of Singapore https://orcid.org/0000-0003-0488-0183 Thorsten Wohland (  twohland@nus.edu.sg ) National University of Singapore https://orcid.org/0000-0002-0148-4321


Introduction
Cell culture is the cultivation of cells derived from an animal or plant source in an arti cially controlled environment. It is a major model system in cell and molecular biology [1] [2]. Fluorescence microscopy of live cells allows investigation of cellular processes in a non-destructive manner. Fluorescence is highly sensitive, and single-molecule detection is possible [3][4] [5].
We detail here a protocol to transfect uorescent proteins and prepare live cell samples for uorescence microscopy. While this protocol uses CHO-K1 cells, it can be used for most adherent cell types with suitable modi cations as required. 2. Warm all media and solutions to 37°C in a water bath before use.

Reagents
3. Culture CHO-K1 (Chinese Hamster Ovary) cells (CCL-61) in DMEM (high glucose with L-glutamine, without sodium pyruvate) supplemented with 1% penicillin-streptomycin and 10% FBS at 37 °C in a 5% (v/v) CO 2 environment. Once the culture is ~90% con uent it can be used for transfection. 4. Remove the spent media from the culture ask and discard. 5. Wash the ask twice with 5 ml PBS (without Ca 2+ and Mg 2+ ).
6. Add 2 ml Trypsin-EDTA (1X) and incubate the ask at 37°C for 5-15 minutes to detach the cells. • Increase the plasmid amount used. Larger plasmids require more amounts to get successfully transfected.
• Optimize the electroporation settings. While the manufacturer provides electroporation parameters for a number of cell lines, this might need to be optimized further. Alternatively, a lipid-based transfection reagent (e.g. Lipofectamine 3000, FugeneHD, etc.) can be tried.
• Cells which are of lower passage number will usually get transfected better. We typically avoid using cells with passage numbers > 20.
2. After transfection, the cells will need time (typically at least 24 hours) to recover their normal morphology and characteristics.
• While we usually use the cells 36-48 hours after transfection for experiments, they can be used anytime from 24 to 72 hours after transfection as long as the cells are healthy and show normal morphology.

Contamination issues:
• Always handle cells in a biosafety cabinet and practice proper aseptic techniques.
• Ensure the equipment, media and other reagents used are sterile.
• Ensure the antibiotic used is not degraded and of correct concentration (Note: good cell culture practice is to avoid using antibiotics in the rst place; if proper aseptic techniques are practiced, antibiotics are not required).

Wash steps with buffers:
• The buffers used for washing prior to trypsinization should not contain Ca 2+ and Mg 2+ as these ions promote cell adhesion.
• Conversely, the buffers used for washing prior to imaging should contain Ca 2+ and Mg 2+ to avoid detachment of cells.

Trypsinization
• While cells will usually detach quickly upon trypsinization, it can take upto 15 minutes depending on the cell type and the potency of the trypsin used. Avoid old trypsin stocks or stocks that have been subjected to temperature changes multiple times.
• Do not use very high trypsin concentrations or leave the cell culture for too long in trypsin as it will kill the cells. If the cells are not detaching easily, a cell scrapper can be used as an alternative.
• The culture media used for neutralizing the trypsin after cell detachment should contain serum. Serum contains protease inhibitors that inhibit the activity of trypsin.

Time Taken
A freshly passaged cell culture would be con uent in 2-3 days depending on the initial number of cells seeded. The electroporation protocol would take about an hour. Post-transfection recovery of the cells would take atleast 24 hours.

Anticipated Results
Fluorescence can be observed in the cells a few hours after transfection. After the cells have recovered post-transfection and regained their normal morphology, they can be used for experiments.