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Cell Culture Media

First you need to find what kind of cell you want to culture. Then you can find what kind of culture media from supplier you gonna need.

The common culture media as following:

  • DMEM (Dulbecco's Modified Eagle Medium)
  • MEM (Minimum Essential Medium)
  • Roswell Park Memorial Institute (RPMI) 1640
  • et al.

For a ready-to-use culture media, you may also need to add Fetal Bovine Serum (FBS) and Antibiotics.

Thawing of Frozen Cell

  • Collect an ampoule of cells from liquid nitrogen storage wearing appropriate personal protective equipment and transfer to the laboratory in a container of liquid nitrogen or on dry ice. (It is important to handle the ampoules with care: on rare occasions ampoules may explode on warming due to expansion of trapped residual liquid nitrogen.)
  • In a microbiological safety cabinet, hold a tissue soaked in 70% alcohol around the cap of the frozen ampoule and turn the cap a quarter turn to release any residual liquid nitrogen that may be trapped. Re-tighten the cap. Quickly transfer the ampoule to a 37°C water bath until only one or two small ice crystals, if any, remain (1-2 minutes). It is important to thaw rapidly to minimize any damage to the cell membranes.
  • Note: Do not totally immerse the ampoule as this may increase the risk of contamination.
  • Wipe ampoule with a tissue soaked in 70% alcohol prior to opening.
  • Pipette the whole content of the ampoule into a sterile tube (e.g. 15 ml capacity). Then slowly add 5ml pre-warmed medium that has already been supplemented with the appropriate constituents. Determine the viable cell density using trypan blue or microscope. Transfer the appropriate volume of cell suspension to a flask to achieve the cell seeding density recommend on the cell line data sheet.
  • For adherent cell lines: Adjust the volume of the medium, and if necessary the flask size, to achieve the cell seeding density recommended on the cell line data sheet. A pre-centrifugation step to remove cryoprotectant is not normally necessary as the first media change will remove residual cryoprotectant. If it is, then this will be specified on the data sheet. If the cells are to be used immediately (e.g. for a cell based assay), rather than subcultured, it may be advisable to perform a pre-centrifugation step to remove cryoprotectant.
  • Incubate at the temperature and CO2 level recommended on the data sheet. If a CO2 fed incubator is used the flask should have a vented cap to allow gaseous exchange.
  • Examine cells microscopically (phase contrast) after 24 hours and sub-culture as necessary.

Resource: some of text from Sigma Aldrich

Cell Passage

If last step is fine, you will see the bottom of the flask is hazy . Observe it with microscope, you'll know the approximate cell density. Here will teach you how to passage cell.

  • Warm media and trypsin in 37 Celsius water bath.
  • Using the pipette, empty liquid media covering cells. Be careful to not touch the pipet to anything outside of the T flask.
  • Add x mL trypsin to T flask. Lightly swish flask, until see some cells leave the bottom of flask.
  • Add y mL culture media. Using the pipette wash all the cell, until them leave the bottom.
  • Pipet mixture out of flask and put in 15 mL centrifuge tube.
  • Centrifuge cells for 4 min at 650 g.
  • Using the pipette, empty liquid media, then add new media y mL in tube.
  • Count cells by microscope, then add cells to a new flask, and add new media.

Resource: some of text from Rice

cell_culture.txt · Last modified: 2020/10/07 06:45 by liuxuan