Cell culture media
Cell culture media kdorfman Tue, 10/11/2011 - 19:24FBS aliquots
FBS aliquots kdorfman Tue, 10/11/2011 - 20:15FBS comes in 500 mL bottles.
Aliquot:
volume (mL) | # aliquots | for |
---|---|---|
50 | 3 | 500 mL DMEM or non-CO2 medium |
37.5 | 4 | 500 mL F10-Ham's |
25 | 4 | 250 mL DMEM or non-CO2 medium |
18.75 | 4 | 250 mL F10 Ham's |
6.25 | 4 | to make freezing media |
2013: Try
- Fisherbrand™ Research Grade Fetal Bovine Serum
- 03-600-511
- 500 mL for $133
Krackeler 45-F0926-500ML $145
Carrie's brand: Atlanta Biologicals Premium S11150 ~$300
Also use Krackeler 12103C ~$314
2019 Genessee GenClone FetalPURE™ Cat #: 25-525 $247.45
Antibiotic/Antimycotic
Antibiotic/Antimycotic kdorfman Tue, 10/11/2011 - 20:21Anti-Anti comes in 100 mL bottles.
Aliquot:
volume (mL) | # aliquots | for |
---|---|---|
10 | 1 | 1 L |
5 | 12 | 500 mL |
2.5 | 12 | 250 mL |
Keep frozen until ready to use.
Fisher SV30079.01, $20
DMEM
DMEM kdorfman Tue, 10/11/2011 - 19:30Medium for 3t3 fibroblasts and B16 cells
bring to final volume, filter sterilize in BSC, refrigerate
- if using DMEM without pyruvate, add 1 mL Na Pyruvate (100mM) per 100 mL medium
Ingredient | supplier | cat # | 1 L | 500 mL | 250 mL | |
---|---|---|---|---|---|---|
DMEM | 13.4 | 6.7 | 3.35 | g | ||
NaHCO3 | 3.7 | 1.85 | 0.925 | g | ||
HEPES | 1.3 | 0.65 | 0.325 | g | ||
FBS | Krackeler | 12103C | 100 | 50 | 25 | mL |
anti/anti | Fisher | SV30079.01 | 10 | 5 | 2.5 | mL |
Mix DMEM, NaHCO3, HEPES in about 70% of the final volume of dH2O.
Initial pH ~7.5
Adjust pH to 7.2 with HCl (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.
F10-Ham's
F10-Ham's kdorfman Tue, 10/11/2011 - 19:49for all LLCPk cell lines
Thaw FBS and anti-anti first!
bring to final volume, filter sterilize in BSC, refrigerate
Ingredient | supplier | cat # | 1 L | 500 mL | 250 mL | |
---|---|---|---|---|---|---|
F10 (Ham's) | Sigma | N6635 | 4.9 | 2.45 | 1.225 | g |
Optimem | Invitrogen | 22600-050 | 6.8 | 3.4 | 1.7 | g |
NaHCO3 | 1.8 | 0.9 | 0.45 | g | ||
HEPES | 0.66 | 0.33 | 0.165 | g | ||
FBS | Krackeler | 12103C | 75 | 37.5 | 18.75 | mL |
anti/anti | Fisher | SV30079.01 | 10 | 5 | 2.5 | mL |
Mix F10, Optimem, NaHCO3, HEPES in about 70% of the final volume of dH2O.
Initial pH ~7.1
Adjust pH to 7.2 (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Refrigerate
For serum free, replace serum with distilled water
Freezing media
Freezing media kdorfman Tue, 10/11/2011 - 20:42Medium with 15% DMSO and 20% serum to protect cells in liquid nitrogen.
F10 Hams for freezing (initial serum concentration = 0.075)
DMEM for freezing (initial serum concentration = 0.1)
Filter sterilize
Non-CO2 Media
Non-CO2 Media kdorfman Tue, 12/27/2011 - 18:37Try this: http://www.thermofisher.com/us/en/home/life-science/cell-culture/mammal…
FluoroBrite™ DMEM Media
serum-free
serum-free kdorfman Wed, 10/19/2011 - 15:07Non-CO2 serum-free medium
For working with live cells at the microscope
bring to final volume, filter sterilize in BSC, refrigerate
Ingredient | supplier | cat # | 1000 | 500 | 250 | mL |
---|---|---|---|---|---|---|
MEM | Sigma | M3024-1L | 13.4 | 6.7 | 3.35 | g |
HEPES | Acros | 172571000 | 1.3 | 0.65 | 0.325 | g |
Na pyruvate 100 mM | Thermo | SH30239.01 | 10 | 5 | 2.5 | mL |
anti/anti | Fisher | SV30079.01 | 10 | 5 | 2.5 | mL |
Mix MEM and HEPES in about 70% of the final volume of dH2O.
Initial pH ~6.3
Adjust pH to 7.3
Add appropriate aliquot of antibiotic/antimycotic, Na pyruvate
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.
with serum
with serum kdorfman Tue, 10/11/2011 - 20:00Non-CO2 Medium
For working with live cells at the microscope, when serum is needed for normal division.
bring to final volume, filter sterilize in BSC, refrigerate
Ingredient | supplier | cat # | 1 L | 500 mL | 250 mL | |
---|---|---|---|---|---|---|
MEM | 13.4 | 6.7 | 3.35 | g | ||
HEPES | 1.3 | 0.65 | 0.325 | g | ||
Na pyruvate 100 mM | 10 | 5 | 2.5 | mL | ||
FBS | Krackeler | 12103C | 100 | 50 | 25 | mL |
anti/anti | Fisher | SV30079.01 | 10 | 5 | 2.5 | mL |
Mix MEM and HEPES in about 70% of the final volume of dH2O.
Initial pH ~6.3
Adjust pH to 7.3
Add appropriate aliquot of FBS, antibiotic/antimycotic, Na pyruvate
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.
Trypsin
Trypsin kdorfman Tue, 01/19/2016 - 18:58Try Fisher 12-605-010
Stable at room temp!
Gibco™ TrypLE Express Enzyme (1X), Phenol Red
Animal origin-free, recombinant enzyme
$20.10 -for 100 mL