Standard Reagents for Cell Culture

Stock reagents

10x PBS	Phosphate-Buffered Saline Storage at RT
80.1 g	NaCl (= 1370 mM)
2.0 g	KCl (= 27 mM)
7.65 g	Na₂HPO₄ * 2 H₂O (= 43 mM)
2.0 g	KH₂PO₄ (= 14.7 mM)
	adjust vol. to 1.000 ml the pH should be 7.4 w/o adjustment divide in aliquots and sterilise by autoclaving

10x PBS-CM (w/ Ca/Mg)	Phosphate-Buffered Saline with Calcium and Magnesium Storage at RT
80.1 g	NaCl (= 1370 mM)
2.0 g	KCl (= 27 mM)
14.24 g	Na₂HPO₄ * 2 H₂O (= 80 mM)
2.7 g	KH₂PO₄ (= 20 mM)
1.47 g	CaCl₂ * 2 H₂O (= 10 mM)
1.23 g	MgSO₄ * 7 H₂O (= 5 mM)
	adjust vol. to 1.000 ml the pH should be 7.4 w/o adjustment divide in aliquots and sterilise by autoclaving

5x Trypsin-EDTA	Storage at 4 °C (for 1 - 2 weeks). Aliquot and store at -20 °C (long term).
0.25 g	trypsin (= 0.25%)
0.02 g	Na₂EDTA * 2 H₂O (= 0.02% = 0.53 mM)
	adjust vol. to 100 ml with 1x PBS and sterilise by filtration (PES or Nylon)

1 M HEPES, pH 7.3	Storage at 4 °C
11.9 g	HEPES, free acid (better would be to use the Na-salt)
40 ml	A. dest.
	adjust to pH 7.3 with NaOH (~ 3 ml 5 M) adjust vol. to 50 ml and sterilise by filtration (PES)

100 mM Dexamethasone stock	Storage at -20 °C
100 mg	Dexamethasone
2.55 ml	EtOH

100 mM PMA stock	Storage at -20 °C
25 mg	Phorbol-12-Myristate-13-Acetate
405 µl	DMSO

5 mM β-Mercaptoethanol stock	Storage at 4 °C
5.3 µl	β-Mercaptoethanol
15 ml	PBS
	steril filtered

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Standard solutions and buffers

PBS	Storage at RT
	dilute 10x PBS 1:10 with sterile A. dest.

DPBS	Storage at RT
	dilute 10x PBS 1:10 with sterile A. dest.
	ATCC #30-2200; Biowest #L0615T; Lonza #17-512Q; Mediatech #21-031; Millipore #BSS-1006-B

Trypsin-EDTA	Storage at 4 °C (for 1-2 weeks). Aliquot and store at -20 °C (long term).
0.25%/0.02%	ATCC #30-2101; Biowest #L0932; Cellgro #25-053-Cl
0.05 %	dilute 2 ml Trypsin-EDTA with 8 ml DPBS (=> 0.05% Trypsin, 0.004% EDTA) Trypsin inactivates rapidly at 37 °C

0.4 % Trypan blue	Storage at RT
0.4 g	trypan blue
	adjust vol. to 100 ml with 1x PBS
	ATCC #30-2402; Biowest #L0990 (0.5%); Lonza #17-942E; HyClone #SV30084.01; Mediatech #25-900-CI

CuSO₄	Storage at RT
500 mg	CuSO₄ * 5 H₂O
20 mg	EDTA
	adjust vol. to 1000 ml with A. dest. and autoclave

Gen stock	Storage at -20 °C
25.0 mg/ml	gentamicin-SO₄^2- in A. dest.
	sterilize by filtration
	add 2 µl/ml medium => 50 µg/ml

100 µM Dexamethasone	Storage at -20 °C
1 µl	Dexamethasone stock (100 mM)
999 µl	A. dest.
	add 0.1 µl/ml medium => 10 nM

100 µM PMA	Storage at -20 °C
1 µl	PMA-stock (100 mM)
999 µl	DMSO or PBS
	add 1 µl/ml medium => 100 nM

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Media

Complete media are usually stored up to 4 weeks at 4 °C, for longer time in aliquots at -20 °C.
Non complete media (i.e. w/o Gln, antibiotics, FBS) can be stored up to 1 year at 4 °C.

pH-buffer

Depending on the NaHCO₃ concentration of the medium, different CO₂ concentrations are necessary in the incubator. In order to obtain a pH 7.3 you need (Nomogram):

NaHCO₃	CO₂	typical medium
3.70 g/l	12 %	DMEM
2.20 g/l	7 %	WME, BME, M199, McCoy
2.00 g/l	7 %	RPMI1640
1.50 g/l	5.0 %	DMEM(ATCC)
1.20 g/l	4.0 %	DMEM/F12, F10, F12
0.85 g/l	2.5 %	MEM
0.35 g/l	1 %	Hanks salts

Usually, the higher the NaHCO₃ (and thus the CO₂) conentration, the higher the proliferation rate can be.
The medium is less dependend on a correct CO₂ concentration, when other buffers, e.g. HEPES (5 - 10 mM), are present.
This is also true for the culture flask: flasks with venting membranes are more sensitve for changes of the CO₂ concentration than flask which can be closed tightly.
Phenol red is used as pH-indicator: red at pH > 7.2, if lower it changes to orange. Changes of the pH within 24 h indicates a wrong pH of the medium, a too high initial cell concentration or a bacterial contamination (medium turbid).

Aminoacids, vitamins, salts

The media contain different concentrations of glucose, aminoacids, vitamins, salts. The osmolarity is between 270 - 340 mOsmol/kg. 1 g NaCl = 34 mOsmol/kg.
Minimal media have the double concentration of aminoacids than the basal media - thus basal media need more freequently a medium change.
They are provided for direct use (1x), as 10x stock solutions which have to be diluted with steril MilliQ water or as powder, which has to be dissolved with steril MilliQ water or has to be filtrated afterwards. Due to a liberation of CO₂ caused by the filtration, the pH of the medium can rise up for 0.2 - 0.3.
To these artifical media has to be added serum (usually FBS) to a final concentration of 2 - 20 %. As this is an important cost factor, minimising this concentration is very desirable.
If antibiotica have to be added, they should be dissolved in steril DPBS.
Sometimes pyruvate, glutamine and/or NaHCO₃ have to be added separately:
- Gln is labil (specially at 37 °C) and frequently has to be added directly before the use to liquid media. Glutaminase hydrolysis Gln to Glu and toxic NH₃ (inhibiting cell proliferation). To avoid this, Gln is sometimes added as dipeptide (L-Alanyl-L-Glutamine).
- NaHCO₃ usually has to be added to powder media.
- High pyruvate concentrations (550 mg/l) let the cells produce enough CO₂ for their needs.
Add to medium to obtain the final concentrations:

Gln (200 mM) Storage at -20 °C		Pyr (100 mM) Storage at 4 °C		NaHCO₃ (7.5 %) Storage at RT		HEPES (1 M) Storage at 4 °C
add ml/l	Final conc. mg/l	add ml/l	Final conc. mg/l	add ml/l	Final conc. mg/l	add ml/l	Final conc. mM
3.4	100	...	...	4.7	350	1	1
5.0	146	...	...	11.3	850	2	2
7.5	219	...	...	16.0	1200	5	5
10.0	293	...	...	29.3	2200	10	10
20.0	585	...	...	49.3	3700	20	20

Storage: Usually these media can be congelated for long term storage. In order to avoid several freeze-thawn cycles, make aliquots, which can be used within 2 - 4 weeks.

Applications

DMEM is a very common medium, some are HEPES-buffered (=> lower NaHCO₃ concentration). The original DMEM has 1 g/l Glc, a very successful modification has 4.5 g/l Glc.
Ham F10 or F12 are adequate for growth of CHO cells with low (F10) or w/o (F12) FBS. Also difficult primary cell lines can grow in this medium with 10 - 20 % FBS.
A mixture of DMEM and F12 (50:50) is frequently used for cultivations w/o serum.
RPMI 1640 is good for lymphocytes using 10 - 20 % FBS.
MEM is adequate for suspension cultures. It has less CaCl₂ than other media and is mainly buffered by phosphate.

Sera

Frequently sera (mainly Fetal Bovine Serum, FBS) are added in concentrations between 2 - 20 %. FBS is a non defined natural product, which can change considerably between different charges. A part of containing necessary growth factors, albumin, attachment factors, they can also contain endotoxins LPS, antibodies, microorganisms (virus, mycoplasma), antibiotics.
Although FBS is expensive, media w/o FBS are not necessarily cheaper, because some cells need in that case growth factors.
ITS supplement (5 µg/ml Insulin, 5 µg/ml Transferrin, 5 ng/ml Na-Selenite, Na₂SeO₃) is useful for many cell lines.
FBS: Blood is obtained from 3 - 7 month old bovine fetuses, coagulated and centrifuged; it should not contain much hemoglobin. Afterwards it is sterilized by filtration (0.1 µm).
In contrast to Newborn Calf Serum (NCS), FBS contains more fructose and less immunoglobulins and complement system proteins.
In the case of inhibitory effects, FBS can be inactivated:
- easily by heating for 30 min at 56 °C; shake softly (avoid formation of foam).
- by γ-radiation with ⁶⁰Co.
As by inactivation sometimes the desired effects diminish, the inactivation can not be suggested as a standard procedure. E.g. if in a cytotoxic study the release of cell LDH is determined, the LDH of the serum should be inactivated before!
Storage: Aliquoted at -20 °C for 1 - 3 years. Thawn up slowly (overnight in refrigerator) and avoid frequent freeze thawn cycles.

Main Components	Storage at 4 °C; long term: -20 °C
Component	DMEM high	DMEM / F12	WME	RPMI 1640
D-glucose (g/l)	4.500	3.150	2.000	2.000 4.500 (ATCC)
NaHCO₃ (g/l)	3.700 1.500 (ATCC)	1.200	2.200	2.000 1.500 (ATCC)
HEPES (mM)	--	15	--	25 10 (ATCC)
L-glutamine (g/l)	0.584	0.365	--	0.300
Pyruvate (g/l)	0.110	0.055	0.025	-- 0.110 (ATCC)
Phenol red (g/l)	0.015	0.008	0.010	0.005
Brand	ATCC #30-2002 Biowest #L0103 #L0104 Gibco #10569 #11995 HyClone #SH30243 Lonza #12-604 Cellgro #10-013 Sigma #6429	Caisson #DFL13 ATCC #30-2002	Caisson #WML01 Gibco #12551-032 Sigma #W4128	Gibco #22400 ATCC 30-2001
w/o NaHCO₃	Biowest #L0070 Gibco #128005 Sigma #7777	Caisson #.	Caisson #. Gibco #. Sigma #.	Gibco #.

DMEM: Dulbecco's Modified Eagle Medium
F12: Ham's F-12
WME: Williams' Medium E
In order to use different media within one incubator (thus obviously only one CO₂-concentration) media are also offered w/o NaHCO₃ to adjust its concentration accordingly.

Medium additives	ATCC	Biowest	Gibco	HyClone	Lonza	Cellgro	Sigma
NaHCO₃ (7.5 %)	--	#L0680	#25080	--	--	#25-035-CI	#S8761
HEPES (1 M)	--	#L0180	#15630	--	#17-737	#25-060-CI	#H0887
L-glutamine (200 mM)	#30-2214	#X0550	#25030	#SH30034	#17-605	#25-005	#G7513
Ala-Gln (200 mM)	#30-2115	#X0551	#35050061	#SH30590	--	#25-015-CI	#G8541
Pyruvate (100 mM)	--	#L0642	#11360	--	#13-115	#25-000-CI	#S8636
ITS (100x) SITE+3 (100x)	--	--	#41400	#SV30085	#17-838	#25-800-CR	#I3146 #S5295
DMSO	#4-X	--	--	#TS-20688	--	#25-950-CQC	--
FBS (100 ml)	#30-2021	#S1650 (MEX)	#10437 (non-US)	#SV30160 (South Am.) #SH30088 (standard)	#14-801E (South Am.)	#35-010-CV (500 ml, regular)	#F0926 (non-US)
100x NEAA (100 ml)	#30-2116	#BioWest (MEX)	#11140-050	#HyClone (standard)	#13-114E	#CellGro	#Sigma

ITS (100x): 1,000 µg/ml Insuline, 550 µg/ml Transferrin, 0.6 µg/ml Selenious acid
SITE+3 (100x): 0.5 µg/ml Selenious acid, 1,000 µg/ml Insuline, 550 µg/ml Transferrin, 200 µg/ml Ethanolamine, 470 µg/ml linoleate, 470 µg/ml oleate and 50 mg/ml BSA
Dexamethasone (Sigma, #D4902): 100 mg Glucocorticoid derivate
BPE Bovine Pituitary Extract (Gibco, #13028-014): 25 mg/2.5 ml
rhEGF recombinant human Epithelial Growth Factor (Gibco, #10450-013): 25 µg

Complete medium

To prepare 50 ml complete medium with standard 10% FBS, which can be stored in sterile Falcon tubes, we use the following additives.

WME-L: Williams' Medium E adapted for Liver cell line
WME-L requires additives for the original 500 ml WME flask:
+ 8.3 mg BPE/500 ml WME → 16 µg/ml final concentration
+ 25 µg rhEGF/500 ml WME → 5 ng/ml final concentration

Medium	DMEM (ATCC)	DMEM/F12 (Caisson)	WME-L (Caisson)
Medium	43.5 ml	45 ml	44 ml
FBS (10% final)	5 ml	5 ml	5 ml
HEPES (10 mM final)	500 µl	--	500 µl
Ala-Gln (2 mM final)	--	--	500 µl
SITE+3 (1x final)	--	--	500 µl
Dexamethasone (10 nM final)	--	--	5 µl

Freezing medium

In order to avoid cell damage due to ice crystall fromation during freezing, cryoprotection like DMSO or glycerol (5-10 %) are added to the medium. As the cryoprotectants, specially DMSO, are toxic to the cells, they are mixed double concetrated to the medium (= 2x Freezing medium) and then added to the normal growth medium containing the cells; afterwards the cells have to be cooled and frozen immediately.
Usually freezing medium has also higher concentrations of FBS (10 - 20 %)
DMSO has to be sterilized by filtration (Nylon) while glycerol can be autoclaved.

2x Freezing medium	Prepare just in time
Cell line	2x Freezing medium		Resuspend (T-75)		Final Freezing medium
HeLa, Caco-2	DMEM/F12, 20 % FBS 10 % DMSO	4.5 ml 0.5 ml	Trypsin/EDTA DMEM/F12, 20 % FBS	1.5 ml 3.5 ml	DMEM/F12: FBS DMSO	20 % 5 %
THP-1	DMEM/F12, 20 % FBS (60 °C) 10 % DMSO	4.5 ml 0.5 ml	Trypsin/EDTA DMEM/F12, 20 % FBS (60 °C)	1.5 ml 3.5 ml	DMEM/F12: FBS (60 °C) DMSO	20 % 5 %

60 °C: Heat inactivation of FBS by heating to 60 °C for 30 min.

Providers:
ATCC: http://www.atcc.org/CulturesandProducts/CellBiology/MediaSeraandReagents/tabid/171/Default.aspx
Biowest = Mayimex: http://www.biowest.net
Gibco = Invitrogen
HyClone = Fisher Scientific: http://www.hyclone.com
Lonza = BioWhittacker: http://www.lonzabio.com/; http://www.lonza.com/group/en.html
http://www.lonza.com/group/en/products_services/products/researchproducts/instructions/cellculture.html
Cellgro = Mediatech: http://www.cellgro.com
Millipore = Chemicon
Sigma-Aldrich: http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-cross-reference.html

100x NEAA (Non-Essential Amino-Acids):
Dissolve in 50 ml A. dest. + sterile filtrate:
Ala: 45 mg
Asn: 75
Asp: 65
Arg: 200 - not standard, but difference between EMEM and DMEM
Pro: 60

Glu: 75 - standard but not necessary as in DMEM present
Gly: 40 - dito
Ser: 50 - dito

Last modified: 04.12.2015