COMPLETE CULTURE MEDIA

| AMNIOSTART | Chromosome medium | Instruction for chromosome analysis | HYBRIDOMA medium | HYBRIDOMED DIF 1000 | MCDB 153 Basal | MCDB 153 Complete | OCTOMED | SEBOMED | PFEK-1 | START V Brain Cell medium | TNB 100 |

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SERUM AND PROTEIN FREE CELL CULTURE MEDIA

Media	Cat No.	Serum/Protein Free	Cells/Cell Lines
Coon's F-12	F0855	serum free if supplemented accordingly	FRTL 5
HybridoMed / DIF 1000	F8035 / 1	serum free	hybridormas
	F8055 / 1
Iscove's	F0465	serum free if supplemented accordingly	lymphocytes
MCDB 153	F8105	serum free	(human) keratinocytes
octomed	F8085	protein free	CHO cells
PFEK-1	F8045	protein free	VERO cells
sebomed	F8215	serum free	(human) sebocytes
Start V	F8075	serum free	neuronal cells
TNB 100	F8023	serum free	neuroblastoma x glioma hybrid cells,
			normal neuronal cells, cytotoxic T-cells.

Note: A trypsin inhibitor (cat. no. L2180) might be required with serum free media!

AMNIOSTART

Storage: -20^OC

Complete medium to grow primary human amniotic fluid cells, harvested during early pregnancy (approx week 12 - 16). Amniostart provides a well balanced formulation of salts, amino acids, vitamins, trace elements, hormones, nucleosides, insulin, transferrin, selenious acid, and growth factors.

Amniostart contains 20% pretested Fetal Bovine. Serum (FBS); gentamycin sulphate serves as protective against bacterial contamination.

Amniostart is available in two different versions, buffered either with HEPES (25 mM) or NaHCO₃ .A CO₂ incubator at 5.5% CO₂ is required, and a humidity of 95%.

Amniostart has a shelf life of 36 months at - 20⁰C. Once thawed refrigerated (at +2 +8^oC) medium should be used within 7 days, repeated thawing and freezing might harm its quality.

	Cat.No.	Unit
Amniostart	F5043	100 ml
Amniostart with 25 mM HEPES	F5143	100 ml

CHROMOSOME MEDIUM – LIQUID

Storage: -20^OC

The complete medium is standardized for chromosome analysis with excellent lot to lot consistancy

Formulation (per 1000 ml)

MEM Joklik with non essential amino acids	850 ml
Fetal Bovine Serum	150 ml
Heparin	25000 E
Penicillin G, sodium salt	75000 E
Streptomycin sulphate	50 mg
Phytohemagglutinin L*	2.5 mg
Ascorbic acid	5 mg
Glutathione (reduced)	5 mg

* for Chromosome medium B

	Cat.No.	Unit
Chromosome Medium A	F5013	100 ml
without phytohemagglutinin (PHA) L
Chromosome Medium B	F5023	100 ml
with phytohemagglutinin (PHA) L

Additives for chromosome analysis:

Storage –20^oC

	Cat.No.	Unit
COLCHICIN (10 μg / ml)	L6211	25 ml
in PBS without Ca^2+ / Mg^2+
for mitosis inhibition
COLCEMIDE (Demecolcine)	L6221	25 ml
10 μg / ml in PBS without Ca^2+ / Mg^2+
for mitosis inhibition
COLCEMIDE (Demecolcine)	L6231	25 ml
10 μg / ml in Hanks' saline
for mitosis inhibition

Safety precautions:

· after contact with skin, wash immediately with plenty of water

· wear suitable, protective clothing

· in case of an accident, seek medical advice immediately

INSTRUCTIONS FOR CHROMOSOME ANALYSIS

Blood samples should be token (sufficient cannular diameter, reduced aspiration), as to ovoid haemolysis.

1. Under appropriate (sterile) conditions, approx. 0.5 ml (25 - 30 drops) of heparinized venous whole blood are added and carefully mixed with 8 ml of Chromosome Medium B (or 8 ml of Chromosome medium A +0.08 ml PHA L (cat no M5030) in a tube. Cultivation time should be 72 hrs at +35 – 37^oC.

2. 0.4 ml of Colchicin/Colcemide (10 μg/ml) (cat.nos. L6211 / L6221) are added (corresponding to 0.48 μg/ml of medium concentration); incubate for an additional 2 hrs.

3. Cells are centrifuged for 5 min at approx. 1000 rpm. Remove supernatant and resuspend cells in 5 ml of hypotonic potassium chloride(0.075M) (cat. no. L6413). Incubate for another 12 min at +35 - +37^oC and centrifuge the culture at 1000 rpm for 10 min.

4. Discard all but 0.25 ml of the supernatant, resuspend cells in the remaining fluid.

5. Add 5 ml of freshly prepared, ice-cold fixative (glacial acetic acid/methanol 1:3) dropwise to the cell suspension white gently moving the flask. Allow resting for 10 min at +2 - +8^oC.

6. Centrifuge at 1000 G for 10 min, remove remaining fixative and resuspend cells in 5 ml ice-cold fixative.

7. Following another centrifugation, cells will become visible as a slightly cloudy suspension (approx. 0.5 - 1 ml). Drop 3 - 5 drops of cell suspension from a 30 cm distance onto a moist, non greasy slide.

8. Air-dried slides con be stained readily using the Orcein or Giemsa staining technique.

HYBRIDOMA MEDIUM – LIQUID

Storage: -20^oC

Optimized for the production of monoclonal antibodies.

Formulation (in mg/l)

Reference:

Peters. J.H et.al. Monocioncil Anlibodies - Preparation and Characterization. Springer [1985]

NaCl	6160	Adenine sulphate	2
KCl	400	Guanine•HCl	0.06
Na2HPO4•7H2O	1210	Hypoxanthine	0.06
NaH2PO4•H2O	28	Thymine	0.06
Ca(NO3)•4H2O	80	Uracil	0.06
MgS04•7H2O	120	Xanthine	0.06
CaCl2	40
D-glucose	1800	ATP•Na2	0.2
Phenol red	12.1	AMP	0.04
NaHCO3	2040	Ascorbic acid	0.01
		Biotin	0.162
L-alanine	5	Calciferol	0.02
L-arginine	171.5	D-Ca-pantothenate	0.202
L-aspartic acid	22	Choline chloride	2.5
L-cysteine•HCl	0.02	Folic acid	0.802
L-cystine	56	Myo-inositol	28.01
L-glutamine	260	Menadione	0.002
L-glutamic acid	69.6	Nicotinic acid	0.005
Glycine	18	Nicotinic acid amide	0.805
L-histidine	15.2	p-amino benzole acid	0.81
L-hydroxyproline	18	Pyridoxal•HCl	0.005
L-isoleucine	44	Pyridoxine•HCl	0.805
L-leucine	52	Riboflavin	0.162
L-lysine-HCl	46	Thiamine•HCl	0.802
L-methionine	15	DL-α-tocopherol phosphate Na2	0.002
L-phenylalanine	17	Vitamin A	0.02
L-proline	24	Cholesterin	0.04
L-serine	29	2-Desoxy-D-Ribose	0.1
L-threonine	22	D-Ribose	0.1
L-tryptophane	6	Vitamin B12	0.004
L-tyrosine	24	Fetal Bovine Serum (tested in hybrid cell culture)	10% (v/v)
L-valine	21
Glutathione	0.81
Sodium acetate	10
Fe(N03)3•9H2O	0.14
Polysorbate 80 VG	4

Different from the original formulation, only 0.05 mg/1 riboflavin are used due to danger of photo oxidation.

	Cat.No.	Unit
Hybridoma medium	F5515	500 ml
without HAT, with L-glutamine,
with 10% FBS

HYBRIDOMED DIF 1000 – LIQUID

- Serum free medium for mammalian cells -

Storage: -20^oC

The medium was developed to effectively grow hybridomas, but is also suitable for other cell lines. It is based on a 1:1 mixture of Iscove's medium and Ham's F-12, supplemented with transferrin, insulin, and a BSA/oleic acid complex.

Myelomas, hybridomas and also other lymphoid and non-lymphoid cell lines are cultivated in Hybridomed and show growth rates comparable to those observed in serum-supplemented media. Successfully cultivated cells include: YAC-1 (mouse T-cell lymphoma), HeLa (human epitheloid carcinoma), BJA-B (human EVB-negative myeloma), BHK₂₁ (syrian hamster kidney) and L-psv 129 (mouse L-fibroblast).The medium is available for both 5 % and 10 % CO₂ atmosphere- For use in bioreactors or to cultivate delicate normal ceils, we recommend cat. no. F 8035 (CO₂ should be 10 % to reach physiological pH values). With incubators adjusted at 5 % CO₂, F8055 is recommended. This medium has a reduced level of NaHCO₃ and contains 3 g/l HEPES additionally.

Formulation (in mg/l)

Reference:

Jager,V., Lehmann, J. and Friedl,P. Cytolechnology 1, 319 [1988]

CaCl2•H2O	131.55	L-alanine	17
CuSO4•5H2O	0.00125	L-arginine•HCl	100
FeSO4•7H2O	0.417	L-asparagine•H2O	40
KCl	277	L-aspartic acid	36
KNO3	0.038	L-cysteine•HCl•H2O	17.5
MgSO4•7H2O	100.04	L-cystine	35
MgCl2•6H2O	61.5	L-glutamic acid	80
NaCl	5800	L-glutamine	438.6
Na2HPO4	54.5	Glycine	30
NaH2PO4•H2O	81.67	L-histidine•HCl•H2O	30
NaSeO3	0.01	L-isoleucine	105
ZnSO4•7H2O	0.431	L-leucine	105
NaHCO3	3610	L-lysine•HCl	120
		L-methionine	36
D-Biotin	0.0101	L-phenylalanine	50
Choline chloride	8.98	L-proline	37.25
Folic acid	2.66	L-serine	52.5
Myo-inositol	12.6	L-threonine	77.25
Nicotinamide	2.0	L-tryptophane	15
D-Ca-pantothentate	2.24	L-tyrosine	52.31
Pyridoxine•HCl	0.031	L-valine	70
Pyridoxal•HCl	2.0
Riboflavin	0.219	Hypoxanthine	2.04
Thiamine•HCl	2.17	Thymidine	0.365
Vitamin B12	0.6865	Linoleic acid	0.042
D-glucose	3650	Putrescine•HCl	0.0805
Na-pyruvate	330	Lipoic acid	0.105
2-aminoethanol	1.2	Phenol red Na	8.586
		Protein supplements:
		Bovine insulin	10
		Bovine transferring (iron saturated)	10
		Bovine serum albumin free of fatty acids	1000
		oleic acid	8

Different from the original formulation, only 0.05 mg/1 riboflavin are used due to avoid negative photo oxidative effects.

	Cat.No.	Unit
HybridoMed DIF 1000	F8035/1	500 ml
with L-glutamine
with NaHCO3
for 10% CO2
HybridoMed DIF 1000	F8055/1	500 ml
with L-glutamine
with NaHCO3 and HEPES
for 5% CO2

MCDB 153 – BASAL MEDIUM

Storage: +2 - +8°C

The defined medium well supports the growth of normal human keratinocytes if supplemented with EGF, insulin, hydrocortisone, ethanolamine, and phospho-ethanolamine. Instead of these. Fetal Bovine Serum (FBS) can serve as a general supplement. A 5 % CO₂ atmosphere is recommended.

Formulation (in mg/1)

Reference.

Boyce, S.T. and Ham, R.G. J. of Invest. Dermatol. 81, 33 [1983]

NaCl	7599	Choline chloride	13.96
KCl	111.83	Putrescine	0.1611
Sodum acetate•3H2O	500	Vitamin B12	4.07
Na2HPO4•7H2O	536.2	Biotin	0.0146
MgCl2•6H2O	122	Calciumpantothenate	0.258
CaCl2•2H2O	4.411	Nicotinamide	0.03663
Glucose	1081	Pyridoxin•HCl	0,06171
Na-pyruvate	55	Thiamine•HCl	0.3373
NaHCO3	1176	Adenine	24.32
Phenol red	1.377	Myo-inositol	18.02
HEPES	6600	Lipoic acid	0.2063
		Thymidine	0.7266
L-alanine	8.91	Folic acid	0.79
L-arginine•HCl	2107	Riboflavin	0.03764
L-asparagine	15.01
L-aspartic acid	3.99	CuSO4•5H2O	0.0002496
L-cysteine•HCl•H2O	42.04	FeSO4•7H2O	1.39
L-glutamine	877.2	MnSQ4•5H2O	0.000241
L-glutamic acid	14.71	(NH4)6Mo7O24•4H2O	0.001236
Glycine	7.51	NiC12•6H2O	0.0001188
L-histidne•HCl•H2O	16.77	H2SeO3	0.003869
L-isoleucine	1.968	Na2SiO3•9H2O	0.1421
L-leucine	65.6	SnCl2•2H2O	0.0001128
L-lysine•HCl	18.27	NH4VO3	0.000585
L-rnethionine	4.476	ZnSO4•7H2O	0.144
L-phenylalanine	4.956
L-proline	34.53
L-serine	63.06
L-threonine	11.91
L-tryptophane	3.06
L-tyrosine	2.718
L-valine	35.13

	Cat.No.	Unit
MCDB 153 liquid medium	F8105	500 ml
without L-glutamine

MCDB 153 – COMPLETE MEDIUM

Storage: <- 20⁰C

The serurn free medium was supplemented ready-to-use, to support the growth of human normal keratinocytes.

Formulation (in mg/l)

NaCl	7599	Choline chloride	13.96
KCl	111.83	Putrescine	0.1611
Sodum acetate•3H2O	500	Vitamin B12	4.07
Na2HPO4•7H2O	536.2	Biotin	0.0146
MgCl2•6H2O	122	Calciumpantothenate	0.258
CaCl2•2H2O	4.411	Nicotinamide	0.03663
Glucose	1081	Pyridoxin•HCl	0,06171
Na-pyruvate	55	Thiamine•HCl	0.3373
NaHCO3	1176	Adenine	24.32
Phenol red	1.377	Myo-inositol	18.02
HEPES	6600	Lipoic acid	0.2063
		Thymidine	0.7266
L-alanine	8.91	Folic acid	0.79
L-arginine•HCl	2107	Riboflavin	0.03764
L-asparagine	15.01
L-aspartic acid	3.99	CuSO4•5H2O	0.0002496
L-cysteine•HCl•H2O	42.04	FeSO4•7H2O	1.39
L-glutamine	877.2	MnSQ4•5H2O	0.000241
L-glutamic acid	14.71	(NH4)6Mo7O24•4H2O	0.001236
Glycine	7.51	NiC12•6H2O	0.0001188
L-histidne•HCl•H2O	16.77	H2SeO3	0.003869
L-isoleucine	1.968	Na2SiO3•9H2O	0.1421
L-leucine	65.6	SnCl2•2H2O	0.0001128
L-lysine•HCl	18.27	NH4VO3	0.000585
L-rnethionine	4.476	ZnSO4•7H2O	0.144
L-phenylalanine	4.956
L-proline	34.53	<rh> Epidermic growth factor	0.005
L-serine	63.06	Bovine Insulin (Zn Salt)	5.0
L-threonine	11.91	Hydrocortisone	0.5
L-tryptophane	3.06	Ethanolamine	6.108
L-tyrosine	2.718	Phosphoethanolamine	4.1
L-valine	35.13

	Cat.No.	Unit
MCDB 153 complete	F8115	500 ml
with L-glutamine
with 1.176 g/l NaHCO3

SEBOMED – LIQUID

- MEDIUM FOR HUMAN SEBOCYTES -

Modified DMEM/HAm's F-12 (1:1), was optimized to grow the human sebaceous gland cell line SZ95 in vitro The cell line itself was developed by transfecting primary human facial sebocytes. A defined medium formulation (in particular of the complete version) allows detailed studies under controlled conditions on the influence of hormones, cytokines, lipids, and drugs on human sebocytes.

The formulation is patent protected, and available on request.

Reference:

Zouboulis Chc et al Establishment and charocterization of an immortalized human sebaceous gland cell line (SZ95) J. Invest Dermatol. 113, 101(1999)

	Cat.No.	Unit
sebomed basal medium	F8205	500 ml
storage: + 2 - + 8 ^oC

For optimal performance, this basal medium needs to be supplemented with 0.1 ng/ml Epidermal Growth Factor (EGF rh; cat. no, W0001 - 500), and 0.1 ml/ml Fetal Bovine Serum. Gentamycin sulphate at a concentration of 50 μg/ml should be used to protect against bacterial contamination.

	Cat.No.	Unit
sebomed complete medium	F8215	500 ml
storage: - 20 ^oC
Basal medium supplemented with
2.5 mM N-acetyl-L-alanyl-L-glutamine
2.0 g/l NaHCO3
50 mg bovine pituitary extract (BPE)
50 μg/l epidermal growth factor (EGF <rh>)
1 g/l fatty acid free bovine serum albumin (BSA)
0.15 μM linoleic acid

PFEK–1 MEDIUM – LIQUID

Storage: +2 - +8^oC

With this serum and protein free medium, VERO cells proliferate without prior adaptation. VERO cells in protein free culture provide a sensitive substrate for the propagation.of human pathogenic viruses. Cells were infected by several viruses (Coxsackie B4, Herpes simplex type 1 and 2, measles, and polio, type 1 to 3). Virus titres were comparable to those obtained in serum-supplemented media (see table). Due to the very high calcium content of the medium, minor precipitates may occasionally be observed. However, this does not influence cell growth rates.

Virus yield in VERO cells grown in protein free PFEK-1 medium, and in medium* containing serum.

Virus titre (TCID₅₀/ml)**

Virus type	VERO-cells, protein free		VERO-cells,
			medium supplemented with serum
	I ***	II ****
Coxsackie B4	6.3 x 10^8	7.8 x 10^8	5.9 x 10^8
HSV-1	2.1 x 10^8	8.9 x 10^7	1.5 x 10^8
HSV-2	3.4 x 10^7	4.1 x 10^7	3.1 x 10^7
Measles	7.9 x 10^5	1.2 x 10^6	9.8 x 10^5
Polio 1	4.3 x 10^8	5.1 x 10^8	6.6 x 10^8
Polio 2	1.2 x 10^8	9.4 x 10^7	4.8 x 10^8
Polio 3	8.5 x 10^7	7.8 x 10^7	9.2 x 10^7

* Cinatl, J.jr., J Cinatl, H.Robenau, J,Rapp, B.Kornhuber, H.-w.Doerr Protein Free.Culture of VERO cells:

A substrate for Replication of Human Pathogenic Viruses, Cell Biology International 17 (9), 885 [1993]

**TCID 50, 50% tissue infective dosis

*** Virus titer obtained without former passage in protein free medum

**** Virus titer after four passages in VERO cells cultivated in PFEK-1 Medium

Formulation (in mg/l)

L-glutamine	292	NaF	0.003
NaCl	7179.5	NaSeO3•5H2O	0.00228
KCl	310	Na2SiO3•9H2O	0.004
Na2HPO4	40	ZnSO4•7H2O	0.5
MgSO4•7H2o	45.5	FeSO4•7H2O	0.4
MgCl2•6H2O	62	Fe(NO3)3•9H2O	0.1
CaCl2•2H2O	486	CuSO4•5H2O	0,0025
MES	3150	CoC12•6H2O	0.001
NaHCO3	2250	BaCl2•2H2O	0.0023
D-glucose	3650	LiCl	2.5
D-ribose	0.2
Na-pyruvate	85	EDTA Na	4.3
Phenol red Na	6.36	Glutathione	0.03
		Guanine•HCl	0.1
D-biotin	0.06	Hypoxanthine	4.1
Calciferol	0.05	Linoleic acid	0.084
D-panthotenate	1.8	DL-lipoic acid	0.65
Choline chloride	7.5	Oxalic acetic acid	3
FAD•Na2	0.015	Progesterol	0.004
Myo-inositol	15	Putrescine•HCl	0.0805
Nicotinamide	4.5	Na2(Fe(CN)5NO)•2H2O	3
Nicotinic acid	0.02	Taurine	20
p-amino benzoic acid	0.02	Thymidine	0.75
Pyridoxal•HCl	1	Polysorbate 80 VG	2.5
Pyridoxin•HCl	0.1	Riboflavin	0.025
Vitamin A acetate	0.08	Folic acid	1.2
Thiamine•HCl	0.3
DL-tocopherol acetate	0.1	Na2MoO4•2H2O	0.00005