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cell_competition_tissue_culture.md

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Cell Competition Tissue Culture Protocol

Author: Nathan J. Day

Date: November 2021

This protocol shows how to set up either a MDCK wild-type vs. RasV12 or wild-type vs. ScrKD cell competition experiment.

Contents:

  • Reagents & consumables
  • Passasing a cell line
  • Preparing a competition experiment for imaging
  • Notes

Reagents & consumables

Cell culture:

Cells T25 flasks Phosphate-Buffered Saline (PBS) Dulbecco's Modified Eagle Media (DMEM), tetracycline (tet) free

Experiments:

24-well glass bottom plates Doxycycline Trypsin

Passaging a cell line

In order to maintain a healthy population of MDCK epithelial cells, they need to be "re-homed" (passaged) in T25 flasks approximately twice a week (dependent on seeding density). This process involves aspirating the cells, washing/rinsing them with PBS, detaching them from the old flask, diluting them in new media and re-seeding them in a new flask.

1. Warm up Trypsin and tet-free DMEM to 37°C in water bath. 
2. In the laminar flow hood, aspirate media from flask and add 3-5ml of PBS to rinse dead cells away. Aspirate PBS and repeat rinsing step.
3. Add 1ml of Trypsin to the flask and return to the incubator.
4. Wait approx 20-30 mins for the trypsin to detach cells. Check on progress by holding flask up to the light to observe if cells appear in suspension. Give the flask a tap on the side to dislodge any cells from the bottom. 
5. When all the cells appear to have detached, return to the laminar flow hood, add 4ml of DMEM to quench trypsin reaction. 
6. Agitate solution to ensure cells are not clumped together. This can be done using pipette to add and then withdraw the DMEM and cell solution approximately 20 times. 
7. Prepare a new flask with fresh DMEM for the next passage of cells:
    7a. If you are culturing wild-type or Scr<sup>KD</sup> cells then 5mL of DMEM is all that is required for a fresh passage.
    7b. If you are culturing Ras<sup>V12</sup> cells (or other lines) then certain selection agents will be needed in the fresh DMEM. For Ras<sup>V12</sup> cells, blasticidin 1/2000 and zeocin 1/1000 are added to 5mL of DMEM (2.5μL blasticidin and 5μL zeocin). 
8. Add a small volume of previously resuspended cells to the fresh media solution. For a passage rate of twice weekly, approximately 250μL of cell/DMEM solution from the old flask should suffice. This step depends heavily on cell type though so it is best decided through experience. 
    8a. If you are planning on imaging induced Scr<sup>KD</sup> cells then you need to add doxycycline at 1/2500 concentration (2μL per T-25 flask) 2 days before splitting for an experiment.
9. Return newly passaged cells in their T-25 flasks to the incubator.

Preparing a competition experiment for imaging

1. Repeat passaging steps as above, ensuring that you keep the previous passage's cells in the old T-25 flasks. 
    1a. If you are planning on imaging induced Scr<sup>KD</sup> cells then you need to add doxycycline at 1/2500 concentration (2μL per 5ml DMEM in T-25 flask) 2 days before splitting for an experiment.
    1b. If you are planning on imaging induced Ras<sup>V12</sup> cells then you need to add doxycycline at 1/2500 concentration (2μL per 5ml DMEM) in the imaging dish.
2. Take 10μL of cell/DMEM solution and add to cell counter to determine the concentration of cells.
    2a. It is good practice to the four furthest corner squares of the cell counter to gauge an accurate representation of cell solution density. 
3. Calculate the number of cells you need to make up 1x10<sup>5</sup>cells/mL seeding density for imaging.
    3a. The best way to do this is to use the `cell_calculations.xlsx` spreadsheet (created by Giulia Vallardi). You can enter the number of cells counted for 4 squares into this spreadsheet to find the correct volume of cell solution and fresh DMEM to combine. 
4. Make new cell solutions at correct seeding density in eppendorfs or falcon tubes, depending on the volume of solution you are creating. 
    4a. If you are inducing the mutation in either the Ras<sup>V12</sup> or Scr<sup>KD</sup> cells then you need to add doxycycline to the new solution at a concentration of 1/2500 (2μL for 5mL). 
5. Seed new cell solution in imaging dish of choice. 1mL of solution per well for the typical 24-well dishes used for incubator scope experiments. 
6. Place the imaging dish in the incubator for 2 hours to allow the cells to settle and form attachments to the dish. After this you can take the cells to begin imaging.

Notes