Cell culture media

Cell culture media kdorfman Tue, 10/11/2011 - 19:24

FBS aliquots

FBS aliquots kdorfman Tue, 10/11/2011 - 20:15

FBS 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 - 20:21

Anti-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 - 20:00

from Kathleen Arcaro

To make mL D5 DMEM with pyruvate, glucose, glutamine
start with mL water
add: g DMEM (Gibco 12800-058/ fisher 12800017)
add: g NaHCO3 if there is none already in the DMEM
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL insulin solution 10 mg/mL in HEPES
add: mL amino acid solution
add: mL cosmic calf serum
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

insulin solution 10 mg/mL in HEPES

amino acid solution

DMEM for Canine Cancer Cells

DMEM

DMEM kdorfman Tue, 10/11/2011 - 19:30

Medium for 3t3 fibroblasts, HeLa, and B16 cells

To make mL DMEM
start with mL water
add: g DMEM*
add: g NaHCO3
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL FBS
add: mL anti/anti

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 - 19:49

for all LLCPk cell lines

Thaw FBS and anti-anti first!

To make mL F10-Ham's
start with mL water
add: g F10 (Hams) (Sigma N6635)
add: g Optimem (Invitrogen 226000-050)
add: g NaHCO3
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL FBS
add: mL anti/anti

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 - 20:42

Medium with 15% DMSO and 20% serum to protect cells in liquid nitrogen.

F10 Hams for freezing (initial serum concentration = 0.075)

To make mL F10-Ham's for freezing
add: mL F-10 Ham's
add: mL serum
add: mL DMSO

DMEM for freezing (initial serum concentration = 0.1)

To make mL DMEM for freezing
add: mL DMEM
add: mL serum
add: mL DMSO

Filter sterilize

McCoy's 5A

McCoy's 5A kdorfman Tue, 09/19/2023 - 15:19

McCoy'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

If you havemL McCoy's
mix withmL FBS
andmL Pen-Strep
for a final volume ofmL McCoy's ready to use

Non-CO2 Media

Non-CO2 Media kdorfman Tue, 12/27/2011 - 18:37

Try 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 - 15:07

Non-CO2 serum-free medium

For working with live cells at the microscope

To make mL non CO2 medium
start with mL water
add: g MEM (Sigma M3024-1L)
add: g HEPES, then pH to 7.3 (initial pH = ~6.3)
add: mL Na pyruvate 100 mM (thermo SH30239.01)
add: mL anti/anti

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 - 20:00

Non-CO2 Medium

For working with live cells at the microscope, when serum is needed for normal division.

To make mL F10-Ham's
start with mL water
add: g MEM (Sigma M3024-1L)
add: g HEPES, then pH to 7.3 (initial pH = ~6.3)
add: mL Na pyruvate 100 mM (thermo SH30239.01)
add: mL FBS
add: mL anti/anti

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 - 18:58

Try 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