Cell Culture
General Procedures
Before you start
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warm up media to room temperature
make sure there are sufficient supplies on the cart
wash your hands
Working in the hood:
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turn blower on and UV light off
open window about 6” (alarm will sound if open too wide)
wipe hood with 70% ethanol
spray and wipe down bottles / racks with 70% ethanol before moving in the hood
turn on the gas and light the burner
desinfect your hands (with or wihout gloves – gloves are not sterile!) with 70% alcohol
open sterile supplies in hood, reseal package before moving back outside
use the burner to flame pasteur pipettes before use and between different flasks/plates
sterile filter any unsterile solution before adding to media: use 25 mm filters for volumes of 10 – 50 ml,
13 mm filters for 2 - 10 ml
label all plates/flasks with cell line, passage number, date, and your initials
When finished:
-
-
turn off gas
plug pipetor into charger
remove all belongings, do not leave media or tubes in the hood
return media and solutions to their appropriate storage !watch expiration dates !:
-20°C freezer (303):
FBS, pen/strep, trypsin, freezing media
4°C cold room (321): culture media, non-essential amino acids, sodium pyruvate solution
wipe hood with 70% ethanol
pull window all the way down
turn blower off and UV light on
make sure outlets are “ON” so pipetor charges
check waste bottle, empty if full, cover bottom with bleach and soap before re-attaching to vacuum
refill supplies on cart, refill spray bottles with 70% ethanol
keep an eye on the CO2 pressure!
Cleaning procedures
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hood is cleaned before and after EACH use
as required:
empty waste flask, cover bottom with bleach before re-attaching to vacuum
autoclave biohazard trash
hood is cleaned thoroughly once a month (see cleaning schedule for procedures)
incubator is cleaned quarterly AND in cases of severe contamination (see cleaning schedule for
procedures)
In case of contamination
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examine plate with microscope, try to identify the type of contamination (fungi, bacteria, maybe type of
bacteria)
throw out contaminated plates / media outside the TC room, preferably, autoclave right away
clean incubator and hood thoroughly (see cleaning schedule for procedures)
to check media for contamination: put 5 ml of media into T-25 flask, place in incubator overnight, check
under microscope the next day
Elke S. Perloff
116100761
2/12/2016
Supplies
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most supplies (culture dishes, media, pipettes, tubes, etc.) come sterile – open them in the hood and
reseal package before moving back outside
water (for the incubator water pan) and pasteur pipettes need to be autoclaved as needed
Culture dishes
growth area (cm2)
culture medium (ml)
60 mm circular dishes
28
3
6-well cluster plates
9.6
2
12-well cluster plates
4.0
1
24-well cluster plates
2.0
0.5
T-25 flasks
25
3-5
T-75 flasks
75
8-15
6-well transwell plates
4.7
1.5 / 2.5 (top/bottom)
12-well transwell plates
1.0
0.5 / 1.5
24-well transwell plates
0.33
0.3 / 0.8
HTS feeder trays
35
Freezing and thawing cells
-
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label 2 ml cryo tubes with cell type, passage number, date and your initials
gently detach cells from plates (scrape or trypsinize, depending on cell type)
count cells
centrifuge at low (~200) rpm for 5 min, discard supernatent
resuspend in freezing media (Gibco# 11101011, or culture media with 10% DMSO) at 0.5 - 1 x 107
cells/ml
aliquot into cryo vials, place vials on wet ice and start freezing procedure within 5 min
cells are frozen slowly at 1°C/min by placing vials in insulated box in a -80°C freezer for 2 hours and
then transferring them to liquid nitrogen storage
cryopreserved cells are fragile!
remove from liquid nitrogen storage and thaw quickly in a 37°C waterbath
plate cells with culture medium containing 20% (instead of 10%) FBS
use 10 to 20 ml of media per 1 ml of frozen cells; should be at least 3 x 105 viable cells/ml
after 24 hours, replace media with regular media to remove the DMSO
Elke S. Perloff
116100761
2/12/2016