Cells: Freezing, Thawing and Culturing

Overview
This protocol describes in general how to culture, freeze, and thaw cells. Each cell line will be somewhat different, and line-specific procedures should
generally be followed. An excellent source for cell culture is the American Type Cell Collection website (ATCC) which has detailed information on many cell
lines.

Protocol
Thawing Cells
(Note: Perform procedures quickly, as frozen cells are in DMSO, which is toxic to the cells)
1.
2.
3.
4.
5.

Place frozen vial of cells in 37C water bath for ~1 minute until cells are almost completely thawed.
Transfer vial contents to a 15 ml conical tube containing 9 mls pre-warmed media
Centrifuge 5 min 1,500 rpm to pellet cells. Discard supernatant
Re-suspend cell pellet in 10 mls of media and transfer to dish or flask.
Place cells in incubator (generally 37C with 5% CO2).

Culturing Cells

Cells are normally kept in an incubator at 37C and 5% CO2.

Standard media is
o 500 mls of DMEM or RP10
o 50 mls of Fetal Bovine Serum (FCS) or Fetal Calf Serum (FCS)
o 5 mls of 100x Penicillin/streptomycin (Pen/Strep)
Prepare media in the sterile hood. Optional: filter sterilize using a vacuum.
Place media in a 37C water bath to preheat the media before use. Only use your own bottle of media, do not share with others. Media will go bad,
especially if it contains added components with short half-lives. Do not use old media.
All cell work should be done in a BSL2 Biosafety Hood. The hood should be cleaned by spraying with 70% ethanol. Air flow should be maximized by
keeping as few items as possible in the hood. Work as far back into the hood as possible. Avoid moving arms in and out of hood. Spray items that
are placed in the hood with 70% ethanol. Avoid moving arms across any open containers, as most contamination occurs by falling off your clothing

Cells: Freezing, Thawing and Culturing

into media culture. Place lids face down after removing them, to avoid particulates floating into the upturned lids. Try to keep all bottles covered as
much as possible. Place everything you will need in the hood up front
Change media every two days. Remove media via a glass pipette with a plastic pipette tip attached using a vacuum pump. Replace media gently
with a handheld pipetteman.

Cell culture plate dimensions and numbers

The following table gives guidelines for amount of media and cell numbers by plate size.

Vessel

Culture area (cm2) Media vol (ml)

Inoculation density
(cell #)*

Trypsin vol (ml)

100 x 20 mm

56-58

10

1e6

1.0

65 x 15 mm

21

4e5

0.5

35 x 10 mm

10

2.5

1.5e5

0.2

6 well plate

9.4

2.0

2e5

0.2

12 well plate

3.8

1.0

8e4

0.1

24 well plate

1.88

0.5

4e4

0.1

48 well plate

0.8

0.35

1.5e4

0.05

96 well plate

0.32

0.2

6e3

0.02

T-150 Flasks

150

30

T-175 Flasks

175

40

Notes:

HEK293T/17 cells are very small, a fully confluent 100 x 20 mm plate can have up to 40 million cells. Seed a 24 well plate wi th
2.5e5 overnight to get an 80 -90% confluent plate the next day.

Generally use between 0.2 and 0.5 ml of media/cm2 of plate size .

Cells: Freezing, Thawing and Culturing