Insulin

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β2-micoglobulin

 

Insulin

Protein Concentration: 5 μM

Cells: Rat Insulinoma Cells

 

 

Cell Culture Protocol:

TO PREPARE THE MEDIUM:

 

·               RPMI 1640 medium with L-glutamine 500 mL supplemented with;

 

 

FINAL CONCENTRATION

VOLUME

heat-inactivated fetal bovine serum (FBS)

10%

50 mL

penicillin| streptomycin

0.1 mg | mL

5 mL

L-glutamine

2 mM

5 mL

D-glucose

1 mg | mL

5 mL

non essential amino acids (NEAA)

0.1 mM

5 mL

Na-pyruvat

1 mM

5 mL

 

TO SPLIT THE CELLS:

 

·               remove the medium;

·               add 10 mL of trypsin;

·               wait 03 minutes;

·               add 10 mL of RPMI medium;

·               centrifuge: 1250 RPMs for 05 minutes;

·               remove the medium;

·               resuspend the remain pellet in 20 mL of RPMI medium;

·               diluted to new cell culture flasks:

 

1:20 dilution: 19 ml medium + 01 mL culture

1:40 dilution: 19.5 ml medium + 0.5 mL culture

1:3 dilution: 13 ml medium + 07 mL culture – when experiment is planned!

 

References:

 

·               Kapurniotu et. al 1998: EUR J BIOCHEM 251: 208-216;

·               Shearman et. Al 1994: PNAS 91: 1470-1474.

 

 

Cell Plating:

 

plate for MTT:

day 01:

cell density: 5 x 105 cell| mL = 5 x 104 cells|well;

add 100 uLs in each well;

incubate for 24 hrs at incubator;

 

DAY 02:

CHANGE THE RPMI MEDIA FOR A FRESH ONE WITH 1% FBS

 

 

 

Protocol:

  • Incubate insulin in 10mM NaAc (pH 3.2) at a concentration of 2 mM for 24 h prior to adding to the cells. The protein is very soluble at this concentration.
  • Dilute this solution to 50 μM in differential media after 24h and add 10 μl of this solution to the cells.
  • Incubate for 24h and do the MTT assay.
  • If you want to do dose dependence with a drug, prepare the dilutions of the drug in the media in 70 μl volume (for 6 replicates). Add 70 μl of 150 μM insulin to the drug solution. Take out 10 μl of media from the plated cells and add 20 μl of the peptide+ drug to the cells (6 replicates). Do MTT/LDH as required.
  • The viability of 15μM insulin is ~50% by MTT assay

 

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