Laboratory Protocol 2 How do pancreatic cells maintain glycemia constant while entry and consumption of glucose in the organism are highly variable? Aims To identify the stimuli that induce insulin secretion To identify the mechanism by which pancreatic cells maintain glycemia constant To identify the mode of secretion of insulin (on/off response, dose-response, linear response ?) Techniques used Cell culture ELISA assay (Enzyme Linked Immuno-Sorbent Assay) for insulin quantification Materials Culture media INS1E medium RPMI 1640 with glucose 10 mmol/L Hepes 50 µM mercaptoethanol 1 mmol/L Na Pyruvate 1mmol/L 5% Fetal Calf Serum Penicillin (100U/mL) /Streptomycin (100µg/mL) INS1E medium without glucose RPMI 1640 without glucose 10 mmol/L Hepes 50 µM mercaptoethanol 1mmol/L Na Pyruvate 1mmol/L 5% Fetal Calf Serum Penicillin (100U/mL) /Streptomycin (100µg/mL) Stock solutions (provided or to prepare if necessary) - Glucose 1 mol/L : (diluted in KRBH and filtrated) 180.16g/mol : 18 g/100mL - Saccharose 1 mol/L : (diluted in KRBH and filtrated) 342.3g/mol : 34.23 g/100mL - Glutamine 100 mmol/L : 5mL Glutamine 200 mmol/L + 5 mL KRBH - dNTP 100 mmol/L - Measurement Buffer (medium totally free of glucose) KRBH : Krebs Ringer Bicarbonate Hepes pH 7.4 135 mmol/L NaCl 3.6 mmol/L KCl 5 mmol/L NaHCO3 0.5 mmol/L NaH2PO4 0.5 mmol/L MgCl2 1.5 mmol/L CaCl2 10 mmol/L Hepes BSA 0.1% (insulin carrier) Stimulation solutions - Glucose 0 mmol/L : 10 mL KRBH - Glucose 2,5 mmol/L : 10 mL KRBH + 25 µL glucose (1 mol/L) - Glucose 5 mmol/L : 10 mL KRBH + 50 µL glucose (1 mol/L) - Glucose 10 mmol/L : 10 mL KRBH+ 100 µL glucose (1 mol/L) - Glucose 20 mmol/L : 10 mL KRBH+ 200 µL glucose (1 mol/L) - Glucose 40 mmol/L : 10 mL KRBH+ 400 µL glucose (1 mol/L) - Saccharose 20 mmol/L : 10 mL KRBH + 200 µL saccharose 1M - Glutamine 5 mmol/ L: 10mL KRBH + 500 µL Glutamine 100 mmol/L - dNTP 1 mmol/L : 10mL KRBH + 100 µL/nucleotide Material for ELISA assay - 96 well flat bottom plates (Nunc) - Capture antibody (AC1): mouse monoclonal antibody directed against insulin/proinsulin (clone 5E4/3) 1 mg/mL - Detection antibody (AC2): biotinylated-mouse monoclonal antibody directed against insulin (Clone D3E7) 1.7 mg/mL - Extravidin conjugated to HRP (stock 5 mg/mL). Prepare a concentration of 5 µg/mL in PBSBSA 1% - Solutions: PBS Tween 0.05% PBS-BSA 2% PBS-BSA 1% - HRP substrate : 1-StepTM Ultra TMB-ELISA (Thermo Scientific) - HCl 1N Protocol for INS1E cell culture General comments - These cells are adherent to the plastic, they are thus easy to manipulate. However pay attention when you change the medium : add the new medium slowly along the wall of the well with the pipette inclined 30-45°C from the horizontal. - Always pre-heat the medium at 37°C before adding it on cells. Glucose withdrawal from the medium - Eliminate the medium and replace it by the medium without glucose during 2h, 1mL/well; - prepare ELISA plates meanwhile: saturate the plates (see below : ELISA step 2); - pre-incubate the cells at 37°C for 30 min in KRBH buffer (this allows getting rid of the 5% fetal calf serum which contains some glucose). Cell stimulation : - Add 300 µL/well of stimulation medium; - incubate 30 min at 37°C; - to stop the incubation : take out 280 µL of the culture supernatant and freeze at – 20°C. Conditions to be tested : 1. Glucose 0 mmol/L 2. Glucose 1 mmol/L 3. Glucose 2.5 mmol/L 4. Glucose 5 mmol/L 5. Glucose 10 mmol/L 6. Glucose 15 mmol/L 7. Glucose 20 mmol/L 8. Glucose 40 mmol/L 9. Saccharose 20 mmol/L 10. glutamine 5 mmol/L 11. dNTP 1mmol/L If students have enough time, stimuli can be combined together to test the possible synergistic/antagonistic effects of amino acids or nucleotides on glucose effects. ELISA assay protocol Enzyme-Linked ImmunoSorbent Assay, also called ELISA, is an immunological technique used to detect and quantify the presence of an antigen in a sample. In a first step, the antigen to be quantified (or the sample containing the antigen to be quantified) is captured to a surface via a capture antibody adsorbed to a plastic surface. In a second step, a detection antibody specific for the antigen binds to the captured antigen. This antibody is linked to an enzyme, and in the final step a substrate of this enzyme is added. The enzyme converts this substrate in a product whose properties allow the quantification of some detectable signal. This signal is directly proportional to the quantity of detection antibody and finally to the amount of antigen bound to the surface (this linear relation is only true for a certain range of antigen concentrations whose limits have to be determined). In the particular case of the formation of a colored product, photometry can be used to determine the amount of antigen in the sample. The steps of the "sandwich" ELISA used to detect insulin (antigen) are as follows : 1. 2. 3. 4. 5. 6. 7. 8. Coat a plastic surface with the capture antibody. Block any non-specific binding sites on the surface. Apply the antigen-containing sample to the plate. Wash the plate, so that unbound antigen is removed. Apply enzyme-linked revelation antibodies that bind specifically to the antigen. Wash the plate, so that the unbound antibodies are removed. Add a chemical substrate which is converted by the enzyme into a colored product. Measure the absorbance of the plate wells to determine the presence and quantity of antigen. The general principle of photometry is based on the absorption of light (absorbance) by molecules present in the solution. The absorbance of a specimen describes how much of the incident light it absorbs (not all photons get absorbed; some are reflected or refracted instead). For most substances, the amount of absorption depends on the wavelength of the light. If we consider a particular molecule that absorbs a specific wavelength (characteristic of a color), absorbance of the solution at that wavelength is correlated to the concentration of the molecule in the solution. Add 50 µL of the capture antibody solution (AC1) per well at a concentration of 5 µg/mL in PBS 1X according to the designed plate map. Leave 2 h at 37°C ; Discard the solution by flicking the plate ; wash three times in PBS-Tween 0.05% (200 µL/well) ; add 50 µL of PBS-BSA 2% per well ; leave 1h at room temperature ; wash twice in PBS-Tween 0.05% (200 µL/well) ; add 50 µL of each stimulated INS1 supernatant (8 conditions) per well ; draw a calibration curve using known concentrations of bovine insulin diluted in 2% PBSBSA. Determine the absorbance range for which there is a linear relation in between insulin concentration and absorbance. Leave overnight at 4°C ; Discard the solution by flicking ; wash three times in PBS-Tween 0.05% (200 µL/well) ; add the biotin-conjugated detection antibody (AC2): 50 µL/well (5 µg/mL final diluted in 1% PBS-BSA) ; incubate 1 h at 37°C ; add HRP-conjugated streptavidin : 50 µL/well (5 µg/mL final diluted in 1% en PBS-BSA); incubate 45 min at 37°C ; flick the plate ; wash four times in 0.05% PBS-Tween (200 µL/well) ; add 50 µL of TMB substrate per well; add 50 µL of 1M HCl per well to block the reaction ; measure the absorbance at 450 nm.