=====Overview=====
MEF cells are an adherent mammalian cell line. The ones that are currently being used are immortalized. These cells grow rapidly and need consistent "splitting," or "subculturing." Typically, adherent mammalian cell lines are to be subcultured when they reach 70-80% confluence. More than this could result in poor cell viability in subsequent subcultures, or genetic changes and deviation of the cell type to another phenotype. To ensure the most accurate results from your experiments, your cell lines should be subcultured before they reach 90% confluence. This protocol will outline how to subculture MEF cells using 24-well plates in addition to a T25 flask. 

=====Inventory=====
==Reusable==
  * 1000µL pipet
  * 200µL pipet
  * Automatic pipettor (5 or 10mL tips)
  * Repeater pipet (10mL tips)
  * Reusable cell counter slide

==Consumed==
  * Flask containing cells to be subcultured
  * 24-well plate
  * Pipet tips (see REUSABLE for which kind)
  * Trypsin aliquot
  * MEF Growth Media (MGM)
  * (Optional) Hank’s buffer OR phosphate-buffered saline (PBS)
  * Trypan blue dye

=====Protocol=====
  - Assess your cell culture flask and cell confluence using the inverted microscope.
  - Retrieve trypsin aliquot from -20*C freezer and place in the heat water bath.
  - Retrieve cell culture plate, 24-well plate, and MGM and place in the BSL-2 hood using proper aseptic technique.
  - Add media to your new cell culture flask and 24-well plate and incubate in the incubator to warm.
  - When trypsin is done thawing, shake a couple of times to mix, and move to BSL-2 hood with proper aseptic technique.
  - Move the cells that are to be subcultured into the hood.
  - Aspirate off media.
  - Add trypsin to the flask. 2mL for T75, 0.75mL for T25 flasks. Essentially, just add enough to cover the surface area of the flask. Move the flask around to make sure the trypsin engulfs the entire area of the flask.
  - Incubate for 5 minutes. This will detach the cells.
  - Assess flask to see if the cells are in the trypsin suspension, you should be able to see them. If you can't, mechanically tap the flask to attempt to dislodge the cells and assess again.
  - Move the flask to the hood. Add 3mL growth media to the flask using the automatic pipette. This will inactivate the trypsin.
  - Move the cell suspension-trypsin-media mixture to a centrifuge tube.
  - Centrifuge at 1400 RPM for 5 minutes.
  - Aspirate off supernatant.
  - (Optional) Wash step: Resuspend in Hank's buffer. Centrifuge again. Aspirate off supernatant.
  - Resuspend in 1mL growth media (repeat 1-3 times to fully redistribute cells. DO NOT SHAKE.)
  - Calculate the cell density using the cell counter in the cell culture room. To do this, retrieve the reusable cell counting slide and trypan blue dye.
   - Calculate the cell density using the cell counter in the cell culture room. To do this, retrieve the reusable cell counting slide and trypan blue dye.
   - Add 10µl trypan blue and 10µl cell suspension into a centrifuge tube. Take 10µl of this suspension and pipet into the reusable cell counter slide.
   - Take to the cell counter and record the cell density of your cell suspension.
  - MEF cells should be seeded at 8000 cells/cm^2. For surface area numbers, see this table: https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html
   - For T25 flask: y mL = (200,000 cells)/(x cells/mL)
   - For 24-well plate: y mL = (15,200 cells)/(x cells/mL)
  - Add the volume of your cell suspension into the pre-warmed flask and well plate. Label the flask accordingly. Check the flask media-cell suspension to ensure that there are cells in the flask.
  - Incubate overnight. Cells should adhere and show spindles in ~4 hours after seeding.

Note: MEF cells are fairly resilient, and if time is of concern to maintain stock cell lines, the steps that include the spinning-off of trypsin can be skipped. However, if subculturing cells for an experiment, do not skip these steps.