国立研究開発法人 医薬基盤・健康・栄養研究所 JCRB細胞バンク

Supplemented growth hormones for the MCDB153 medium. 

Yasumoto,S. Kanagawa Cancer Center Research Institute. 

[Supplemented growth hormones for the MCDB153 medium.]

5 ug/ml Inslin(1% CH3COOH-DDW) 
0.5ug/ml Hidrocortisone(EtOH) 
10ug/ml transferrin(PBS) 
0.1mM phosphorylethanolamine(DDW) 
0.1mM ethanolamine(DDW) 
10ng/ml EGF(0.1% BSA-PBS) 
40-70ug/ml BPE(PBS) 

[Reference]
 
Jikken Igaku, Supplement
 BioManual Up series (2nd Edition)
 "New Cell Culture methods for Molecular Biology"
 T.Kuroki, N.Hoh and K.Chida
 Yohdo-sha,Co.
 Section 4. Primary cell culture, 2.Keratinocyte P71-80

[Medium]
MCDB153medium Powder (SIGMA cat.No.M-7403) for 1L and adjust to pH7.5 by adding Sodium Bicarbonate 1.176g(1N-NaOH).

MCDB153medium liquid (Functional Peptide Laboratory Inc., IFP) 1L and adust pH to 7.5 by adding stock solution of the sodium bicarbonate (x100-x1000).

[Working solution]
 
Store the working solution in the refregerator up to one month when supplements were added to the medium and only two weeks can be stored in the refregerator when BPE was added.

-- Medium -- 
MCDB153medium liquid (Functional Peptide Laboratory Inc., IFP) 1L and adjust pH to 7.5 by adding stock solution of the sodium bicarbonate (x100-x1000). 

-- Supplement--
Insulin(SIGMA cat.No.I-1882): 5mg/L (1000 x stock solution is prepared in 1% CH3COOH)
Hydrocortisone (SIGMA cat.No.H-4001): 0.5mg/L (10000 x stock solution is prepared in EtOH) 
Transferin (SIGMA cat.No.T8158): 10mg/L (1000 x stock solution is prepared in PBS)  
Ethanolamine (SIGMA cat.No.E-0135): 6.1mg/L (6 ul/L) 
Phosohorylethanolamine (SIGMA P-0503): 14.1mg/L 
EGF (SIGMA cat.No.E-4127): 10ug/L  (1000 x stock solution is prepared in 0.1% BSA-PBS )
BPE(Kyokuto Pherm. Co. cat.No.202000): 40-70mg/L (100 x stock solution is prepared in PBS)  

[Subculture] 

Trypsin solution: Trypsin typeIII(SIGMA cat.No.T-8253) 0.1%  0.02% EDTA in PBS- 
(SIGMA T-8253 was discontinued. Instead, Fujifilm Wako Cat. # 201-19181 will be adequate. Conventional 0.25% trypsin solution will also work.)
Trypsin Inhibitor solution: Trypsin Inhibitor(SIGMA cat.No.T-6522) 2mg/ml in PBS- 
 


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