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
DC5 (DMEM for canine cancer)
DC5 (DMEM for canine cancer) kdorfman Thu, 08/17/2023 - 20:00from Kathleen Arcaro
bring to final volume, filter sterilize in BSC, refrigerate
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
McCoy's 5A
McCoy's 5A kdorfman Tue, 09/19/2023 - 15:19McCoy's 5A (modified) (Fisher 16600-082)
For HCT116 cells (a human colorectal carcinoma cell line initiated from an adult male. The cells are adherent with an epithelial morphology. Following implantation into immunocompromised mice, the cells form primary tumors and distant metastases. In vitro, HCT116 cells grow with a doubling time of about 18 hours.)
Drew's recipe:
10% FBS, 1% Pen-strep
- 500 mL McCoy
- 55 mL FBS
- 5.5 mL pen-strep
OR:
- remove 55 mL from the 500 mL McCoy's from Fisher
- add 50 mL FBS
- add 5 mL pen strep (e.g., MP 1670246)
- resterilize
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