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

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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

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

Medium for 3t3 fibroblasts 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.

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

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

Try this: http://www.thermofisher.com/us/en/home/life-science/cell-culture/mammal…

FluoroBrite™ DMEM Media

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