In-vitro GLP-1 Release Assay Using STC-1 Cells

Materials and Reagents

1. 10 ml Glass bottles
2. 50 ml Glass beaker
3. 5 ml Glass pipettes (Corning, catalog number: 7077-5N )
4. Capillary Micropipettes (Drummond Scientific Company, catalog number: 5-000-2005 )
5. 6-cm dishes (Corning, catalog number: 430166 )
6. 10 ml and 50 ml sterile plastic pipettes with filter (Corning, catalog numbers: 4488 , 4490 )
7. Sterile cell lifts (Corning, catalog number: 3008 )
8. 1.5 ml microcentrifuge tubes (Fisherbrand, catalog number: 05-408-129 )
9. 1.5 ml disposable cuvettes (BRAND, catalog number: 759076D )
10. STC-1 cell line (ATCC, catalog number: CRL-3254^TM)
11. Phosphate-buffered saline (Sigma, catalog number: D8537 )
12. Trypsin (Corning, catalog number: 25-053-CI )
13. DMEM, High Glucose (Hyclone, catalog number: SH30022.02 )
14. Fetal Bovine Serum (Gibco, catalog number: 35050-061 )
15. Enrofloxacin (Sigma, catalog number: 17849-5G-F )
16. Docosahexaenoic acid, DHA (Nu-chek-policy, U-84-A DHA, 1GM), DHA comes as a solution
17. Pure ethanol (Pharmco Products, catalog number: 111000200C1GL )
18. Nitrogen gas
19. HEPES (Sigma, catalog number: H3375 )
20. NaCl (Fisher Scientific, catalog number: BP358-10 )
21. KCl (Sigma, catalog number: P9333 )
22. CaCl₂ (Sigma, catalog number: C1016 )
23. MgCl₂ (Sigma, catalog number: M8266 )
24. EDTA (OmniPur, catalog number: 4050 )
25. Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
26. Tris-base (Fisher Scientific, catalog number: BP152-5 )
27.  PMSF (MP biomedicals, catalog number: 195381 )
28. Halt^TM Protease and Phosphatase Inhibitor Cocktail (100x) (Thermo Fisher Scientific, catalog number: 78444 )
29. Sterile water
30. Protein assay dye reagent concentrate (Bio-Rad, catalog number: 5000006 )
31. High sensitive glugagon-like peptide(GLP-1) active chemiluminescent ELISA kit (Millipore, catalog number: EZGLPHS-35K )
32. 10 mg/ml DHA stock solution (see Recipes)
33. HEPES buffer (see Recipes)
34. 100 µM DHA Stimulation buffer (see Recipes)
35. Cell lysis buffer (see Recipes)
36. 100 mM PMSF stock solution (see Recipes)
    

Equipment

1. Portable Pipet-Aid^® XP Pipette Controller (Drummond Scientific Company, catalog number: 4-000-101 )
2. Tissue culture incubator
3. Refrigerated microcentrifuge (Eppendorf, model: 5424R )
4. Sonicator (Branson 250 Sonifier)
5. ELISA plate reader (TECAN, Sunrise absorbance microplate reader)
   

Procedure

1. Prepare 10 mg/ml docosahexaenoic acid (DHA) stock solution (see Recipe)
   1.  Dilute DHA solution with pure ethanol at 10 mg/ml.
      Note: DHA will stick to plastic material. Always remember to use glass pipettes when you work with fatty acids.
   2. Aliquot DHA stock solution into small glass bottles, 10 ml each.
   3. Ambient air above the solution should be displaced to avoid oxidation of DHA by priming pure nitrogen gas on the surface of the solution prior to closing the container.
   4. Store at -20 °C.
      
   
   
2.  DHA Stimulation assay
   1. Seed 2 x 10⁶ STC-1 cells in 6-cm dishes two days prior to experiment in duplicates. Culture the cells in tissue culture incubator at 37 °C, 5% CO₂, using DMEM with 10% FBS and 20 ng/ml enrofloxacin.
   2. On experimental day cells should be 80% confluent.
   3. Aspirate media and wash the cells twice with 3 ml of HEPES buffer.
   4. Add 3 ml of HEPES buffer into each dish.
   5. Put cells back to tissue incubator for 30 min. The lack of nutrients in the buffer is meant to serve as a ‘starvation’ period prior to nutrient stimulation.
   6. Prepare 100 µM DHA stimulation buffer.
      1. To prepare 20 ml of 100 µM DHA stimulation buffer, add 66 µl of 10 mg/ml DHA (30.4 mM) stock solution into a glass beaker using glass capillary micropipettes. Then add 20 ml of HEPES buffer.
      2. Sonicate the solution immediately at output level 5 and constant duty cycle for 3 min prior to stimulation to prevent DHA precipitation. If using a different sonicator, set the amplitude as 40%. After sonication, the solution should be clear, with minimal bubbling.
         Note: Always prepare this DHA solution freshly, just prior to use.
   7. Aspirate HEPES buffer gently. Then add 1 ml of HEPES buffer into each control dish and 1 ml of DHA stimulation buffer into each stimulation dish. Put all dishes back to tissue culture incubator for 15 min.
   8. Collect 600 µl of media from each dish and put into a fresh 1.5 ml microcentrifuge tube, then place them on ice.
   9. Add 0.6 µl of 100 mM PMSF (see Recipe) into the 600 µl of media at final concentration of 100 µM.
   10. Spin the microcentrifuge tubes at 850 x g for 5 min, at 4 °C.
   11. Transfer 500 µl of media into a fresh tube.
       Note: Samples are now ready for ELISA measurement. One can also store samples at -80 °C up to two weeks.
   12. Measure GLP-1 concentration in samples by ELISA kit according to manufacturer’s instructions. Make sure to set the standard as suggested by the manufacturer.
       
       
3. Lyse the cells in the culture and measure protein concentration from all dishes by Bradford assay   Note: This is meant to account for differences in the number of cells in each dish in these experiments.
   
   1. Carefully remove any residual HEPES buffer in each dish.
   2. Add 1 ml of cold PBS into each dish.
   3. Scrape cells down very carefully using a cell lift. 
   4. Collect cells into a 1.5 ml microcentrifuge tube and put on ice.
   5.  Spin the tubes at 850 x g for 5 min, at 4 °C.
   6. Aspirate supernatants, then add 50 µl of cell lysis buffer and pipette up and down gently to homogenize. Put tubes on ice.
   7. Lyse the cells for 20 min. Vortex every 5 min. 
   8. Spin the tubes at 21,130 x g for 15 min, at 4 °C.
   9. Transfer supernatants into a fresh tube.
   10.  Measure protein concentration using protein assay dye reagent concentrate according to manufacturer’s instructions.

Data analysis

Calculate measured GLP-1 concentration of each sample appropriately from three independent experiments, duplicates in each experiment. Then normalize the value to its corresponding protein concentration value as measured by Bradford assay (Table 1). Use any software package with 4-parameter logistic regression for ELISA analysis.

Table 1. Adjusted GLP-1 concentration

Notes

Using healthy cells is critical for experiments to work. One should check cell growth status and mycoplasma routinely. Always remember to fill DHA stock solution with nitrogen and prepare stimulation buffer fresh prior to use. If one freezes samples at -80 °C, do not freeze and thaw repeatedly.

Recipes

1. 10 mg/ml DHA stock solution
1. 600 µl of DHA liquid coming from the manufacturer (1 g, 1.2 ml in total)
2. 49.4 ml of 100% pure ethanol
3. Aliquot into small glass bottles and fill with nitrogen gas. Store at -20 °C
2. HEPES buffer
   20 mM HEPES, pH 7.9
   140 mM NaCl
   4.5 mM KCl
   1.2 mM CaCl₂
   1.2 mM MgCl₂
3. 100 µM DHA Stimulation buffer
   66 µl of 10 mg/ml DHA stock solution
   20 ml of HEPES buffer
   Sonicate the solution at output level 5 constant duty cycle for 3 min prior to use
4.  Cell lysis buffer
   25 mM HEPES, pH 7.9
   100 mM NaCl
   1 mM EDTA
   10% Glycerol
   1% Triton X-100
5. 100 mM PMSF stock solution
   174.2 mg PMSF
   10 ml of 100% ethanol
   

Acknowledgements

This work was made possible by funding to LS-D from the following sources: NIH 5 K12 HD-068369–05 and Children’s Health Clinical Research Advisory Committee: CCRAC 195.
  This protocol was adapted from Hirasawa et al. (2005).

Competing interests

The authors declare no competing interests.

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