Insulin
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Insulin<o:p></o:p>
Protein Concentration: 5 μM<o:p></o:p>
Cells: Rat Insulinoma Cells<o:p></o:p>
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Cell Culture Protocol:<o:p></o:p>
TO PREPARE THE MEDIUM:<o:p></o:p>
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<![if !supportLists]>· <![endif]>RPMI 1640 medium with L-glutamine 500 mL supplemented with;<o:p></o:p>
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FINAL CONCENTRATION<o:p></o:p> |
VOLUME<o:p></o:p> |
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heat-inactivated fetal bovine serum (FBS)<o:p></o:p> |
10%<o:p></o:p> |
50 mL<o:p></o:p> |
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penicillin| streptomycin<o:p></o:p> |
0.1 mg | mL<o:p></o:p> |
5 mL<o:p></o:p> |
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L-glutamine<o:p></o:p> |
2 mM<o:p></o:p> |
5 mL<o:p></o:p> |
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D-glucose<o:p></o:p> |
1 mg | mL<o:p></o:p> |
5 mL<o:p></o:p> |
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non essential amino acids (NEAA)<o:p></o:p> |
0.1 mM<o:p></o:p> |
5 mL<o:p></o:p> |
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Na-pyruvat<o:p></o:p> |
1 mM<o:p></o:p> |
5 mL<o:p></o:p> |
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TO SPLIT THE CELLS:<o:p></o:p>
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<![if !supportLists]>· <![endif]>remove the medium;<o:p></o:p>
<![if !supportLists]>· <![endif]>add 10 mL of trypsin;<o:p></o:p>
<![if !supportLists]>· <![endif]>wait 03 minutes;<o:p></o:p>
<![if !supportLists]>· <![endif]>add 10 mL of RPMI medium;<o:p></o:p>
<![if !supportLists]>· <![endif]>centrifuge: 1250 RPMs for 05 minutes;<o:p></o:p>
<![if !supportLists]>· <![endif]>remove the medium;<o:p></o:p>
<![if !supportLists]>· <![endif]>resuspend the remain pellet in 20 mL of RPMI medium;<o:p></o:p>
<![if !supportLists]>· <![endif]>diluted to new cell culture flasks:<o:p></o:p>
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1:20 dilution: 19 ml medium + 01 mL culture<o:p></o:p>
1:40 dilution: 19.5 ml medium + 0.5 mL culture<o:p></o:p>
1:3 dilution: 13 ml medium + 07 mL culture – when experiment is planned!<o:p></o:p>
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References:<o:p></o:p>
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<![if !supportLists]>· <![endif]>Kapurniotu et. al 1998: EUR J BIOCHEM 251: 208-216;<o:p></o:p>
<![if !supportLists]>· <![endif]>Shearman et. Al 1994: PNAS 91: 1470-1474.<o:p></o:p>
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Cell Plating:<o:p></o:p>
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plate for MTT:<o:p></o:p>
day 01:<o:p></o:p>
cell density: 5 x 105 cell| mL = 5 x 104 cells|well;<o:p></o:p>
add 100 uLs in each well;<o:p></o:p>
incubate for 24 hrs at incubator;<o:p></o:p>
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DAY 02:<o:p></o:p>
CHANGE THE RPMI MEDIA FOR A FRESH ONE WITH 1% FBS<o:p></o:p>
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Protocol: <o:p></o:p>
- 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. <o:p></o:p>
- Dilute this solution to 50 μM in differential media after 24h and add 10 μl of this solution to the cells. <o:p></o:p>
- Incubate for 24h and do the MTT assay.<o:p></o:p>
- 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.<o:p></o:p>
- The viability of 15μM insulin is ~50% by MTT assay<o:p></o:p>
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