Materials required

 

1.    12 well microtiter plate
2.    0.1 M Krebs-Ringer-Phosphate (KRP) buffer (pH:- 7.2- 7.4)
3.    0.1% SDS
4.    Serum free DMEM
5.    Insulin(100nM) & Plant extracts(100µg/ml)
6.    Radioactive cocktail (10µM 2-deoxy glucose and 0.25µCi of 2 deoxy-D-(3H) – glucose in 12 ml of KRP buffer)
7.    1.5 ml eppendorf tube
8.    15 ml centrifuge tubes
9.    Liquid Scintillation Counter
10.  Pipettes

 

Procedure

 

1.    Take out the differentiated 3T3 L1 cell plate from CO₂ incubator.
2.    Remove the medium from the 12 well plate using a suction pump.
3.    Add 1000 µl serum free media into all wells of the 12 well plate.
4.    Serum starves the cells for 2 hours in CO₂ incubator.
5.    Remove the media using a suction pump.
6.    Add Basal to the wells 1A, 1B and 1C.
7.    Add Insulin to the wells 2A, 2B and 2C.
8.    Add plant extract X and Y to 3A, 3B, 3C, 4A, 4B and 4C respectively.
9.    Incubate the plate at 370C for 30 minutes.
10.  Take out the plate and remove the media.
11.   Add 1000 µl KRP buffer (in room temperature) into all wells and wash cells.
12.   Remove the KRP buffer completely.
13.   Remove the trace amount of KRP buffer also using a pipette.
14.   Add 1000 µl radioactive cocktail into each wells.
15.   Incubate the plate for 5 minutes in a CO₂ incubator.
16.   Discard the cocktail and wash twice with 1000 µl ice cold KRP buffer.
17.   Add 1000 µl of 0.1% SDS to all wells to lyse the cells.
18.   Then scrap in each well using a cell scraper.
19.   Take out the whole cell lysate from each well and add to corresponding labeled scintillation tubes contains 4 ml scintillation fluid.
20.   Mix the tubes gently and keep it for 1-2 minutes to settle all the fluids.
21.   Then measure the glucose uptake rate using the Liquid Scintillation counter.

 

Reagent Preparation

 

0.1 M Krebs-Ringer-Phosphate( KRP) buffer (pH:- 7.2-7.4)

 

Reagents required for making KRP buffer:

 

• NaCl 1.5 M
87.66 g NaCl in 1 Litre deionized water.

• Phosphate buffer pH 7.4:
A) 0.1 M NaH₂PO₄. 2H₂O (MW 156) - 15.6 g in 1 Litre.
B) 0.1 M Na₂HPO₄. 2H₂O (MW 178) - 17.8 g in 1 Litre.
Add 19 ml of A to 81 ml of B, adjust pH to 7.4.

• KCl 0.15 M
5.6 g KCl (MW 74.56) in 500 ml distilled water.

• MgSO₄. 0.15 M
3.7 g MgSO₄.7H₂O (MW 246.48) in 100 ml distilled water.
 
• CaCl₂ 0.11 M
1.62 g CaCl₂.2H₂O (MW 147.02) in 100 ml distilled water.
1.    Make up 100 ml of 1.5 M NaCl to 1 litre.
2.    Add: 120ml of phosphate buffer pH 7.4, 40 ml of 0.15 M KCl, 10 ml of 0.15 M MgSO₄, 10 ml of 0.11 M CaCl₂ – stir carefully to avoid precipitation.
3.    Adjust pH with NaOH (1 M)

 

Radioactive Cocktail

 

 10µM 2-deoxy glucose and 0.25µCi of 2 deoxy-D-(3H) – glucose in 12 ml of KRP buffer.

 

10nM Insulin (Working Standard)

 

10µl of 10µM insulin (Stock) is added into 990µl of serum free DMEM.