Revised by NG Aug 2006
In vitro analysis of THC present in primary adipocytes
1.) Extraction and culture of adipocytes
Extraction
1. Remove epididymal fat pads and place it in 10 mL Krebs (Place in 50 mL falcon
tube).
2. Weigh tissue and place into 50 ml falcon tube
+ 1.5mg Type I collagenase/per gram tissue
+ 2mL Krebs/per gram of tissue
3. Chop up tissue well in falcon tube.
4. Digest tissue at 37°C, shaking speed of 60 cycles/min (40 mins)
5. Dilute digested tissue with 0.5 ml Hepes phosphate buffer/per gram of tissue.
6. Filter diluted tissue through sieve into a 50 ml falcon tube (fat will appear as a
layer on top of soln).
7. Add 10 ml Krebs into 3 x 15 ml falcon tubes.
8. Wash step: Using a cut Pasteur pipette take up fat layer and place into 1st 15 ml
falcon tube, allow the fat to form a layer and repeat for the remaining two tubes.
9. Make up a 100μL mixture of adipocytes:Krebs (1:9) and perform a cell count to
determine the cell density.
Revised by NG Aug 2006
2.) Treating adipocytes in primary culture with Adrenaline
1.) Fill each sample tubes with packed adipocytes (60,000 cells/1.5mL) and top up
each tube with DMEM.
2.) Probe cells with adrenaline (2.75uL) or water.
3.) Incubate for 90 minutes but remember to invert every 15 minutes for 60 minutes.
The last 30 minutes allow cells to settle.
4.) Spin down cells at 4°C for 10 minutes at 12,000 rpm. Transfer supernatant to a
separate sample tube.
5.) Take 50uL of serum for glycerol analysis.
6.) The remainder of the supernatant is used for THC ELISA (10uL) and GC-MS
analysis.
THC ELISA KIT
1. Add 20ul of calibrators, stds and specimens to each well in duplicate.
2. Add 100ul of the enzyme conjugate to each well. Tap sides of plate holder to
ensure proper mixing. Do this in the dark.
3. Incubate for 60 mins at room temp (20-25°C) away from direct light..
4. Wash 6 times with 350ul of distilled water and make sure not to crosscontaminate each well.
Revised by NG Aug 2006
5. Invert wells and vigorously slap dry the wells to ensure that no moisture is left.
6. Add 100ul of substrate reagent to each well and tap sides to ensure mixing.
7. incubate for 30 mins in the dark at room temp.
8. Add 100ul of stop solution to each well to change from blue to yellow.
9. Measure absorbance at 450nm and 650nm and compare average absorbance
obtained from each unknown specimen with the average absorbance of the
positive reference std.
10. Wells should be measured within 1 hour of yellow color development.
Glycerol detection assay
1.) 1 hour prior to the assay, prepare the glycerol standards (see product notes) by
reconstituting glycerol reagent A first and remember to keep this at room temp.
2.) Load 50ul/well of sample to the 96 well plate(s).
3.) Add 50ul/well of reconstituted glycerol reagent A to the plate (including the
glycerol stds at 50ul/well).
4.) Incubate at 25°C for 15 minutes. Pop the bubbles in each well.
5.) Measure the density at 540nm using a plate reader.
Refer to product notes for more details.