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General Protocol

Cell Maintenance Protocol

STC-1 cells should be cultured in supplemented DMEM media (DMEM containing 4.5 g/l D-glucose, without sodium pyruvate) (GlutaMAX, GIBCO, Paisley, UK) with 17.5 % foetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/l streptomycin and incubated in a 5 % CO2 humidified atmosphere at 37 °C. Cells should be passaged at 80–90 % confluence. Passage number should be between 15 and 40.

Experimental Protocol for Test Compounds

Test compounds should be prepared in pre-warmed buffer. Buffer should be tested to ensure compatibility with the particular immunoassay.

Fig. 19.1 Effect of various incubation buffers on levels of Total GLP-1 secreted from STC-1 cells. STC-1 cells (2 × 106) were challenged with mixed nutrients [glutamine + valine + lysine + glycine + glucose + fructose (all at 40 mM)] for 3 h. Samples were prepared in PBS or HANKS or KREBS or HEPES buffers or DMEM media (GlutaMAX, GIBCO, Paisley, UK). The composition of the buffers are as follows; PBS (136.9 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4), HANKS (136.9 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 1 mM MgSO4, 0.2 mM NaHPO4, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 5.6 mM glucose), KREBS (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.25 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 11 mM glucose) and HEPES (140 mM NaCl, 4.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 20 mM HEPES). Total GLP-1 was measured by a specific GLP-1 radioimmunoassay. Statistical comparisons (unpaired Student's t-test) were only performed on 3 h incubations with or without nutrient challenge for each selected buffer. Significant differences are indicated on the graph (*P < 0.05)

Figure 19.1 indicates that dramatically different results can be obtained depending on the buffer system used. Use of buffer solutions which are highly stimulatory (e.g. DMEM media which contains amino acids and vitamins), should be avoided because hormone levels for vehicle controls will be greatly elevated and can distort results. In previous experiments, we have favoured KREBS buffer. The test solution pH should be adjusted to 7.0–7.4 and filter sterilised (0.45 μm filter). If solubility of test compounds is an issue (e.g. in the case of lipids or fatty acids) careful monitoring is needed to ensure test compounds remain in solution for the duration of experiments. To measure acute hormone secretion, STC-1 cells should be seeded into 6 well plates at 1.5 × 106 cells in supplemented DMEM media. After 18 h at 37 °C in 5 % CO2, media should be aspirated and cell monolayers washed 1–2 times with selected buffer. The cells should be acclimatised in buffer for 1 h. Culture media and wash buffers can be kept to ensure that cells have reached basal levels of hormone secretion (Fig. 19.1). After 1 h, the buffer is aspirated and 1 ml test solution added to wells. Cells are then incubated for 1–4 h at 37 °C, 5 % CO2 (in our experience a period of 3–4 h is optimal for hormone secretion, Fig. 19.2). Positive controls known to stimulate gut hormone secretion and negative controls (buffer alone)

Fig. 19.2 Determining the optimal incubation time to measure levels of Total GLP-1 secreted from STC-1 cells. STC-1 cells (2 × 106) were exposed for various incubation times with stimulatory amino acid solution (40 mM glutamine, 40 mM valine, 40 mM lysine and 40 mM glycine) and monosaccharide solution (40 mM glucose and 40 mM fructose) which were prepared in HEPES buffer. Data were statistically compared using the unpaired Student's t-test and significant differences to vehicle controls are indicated *P < 0.05, **P < 0.01 and ***P < 0.001

should always be included in each experimental unit. Post-incubation, we recommend the addition of 10 μl 10X Halt Protease and Phosphatase Inhibitor (Thermo Fisher Scientific, USA) to protect against gut hormone degradation. Cellular supernatants are collected and store at −80 °C prior to further analysis. It is best practice to immediately transfer solutions to tubes placed on ice and to centrifugate (900×g for 5 min) to remove cellular debris. At least three biological replicates should be performed.

 
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