Measurement of GLP-1 release in cell supernatant from Hutu-80 enteroendocrine cells via ELISA 

Plate cells in sterile 24-well plates at 1.5x10^5 per well and leave in a 37°C incubator with stable CO2 conditions overnight.

Check cells are healthy under a microscope.

In a laminar flow hood aspirate medium and wash with PBS 3 times, adding 500µL each time.

Once the final PBS wash is complete add in 500µL of the test media made up for your desired experiment (e.g., high glucose media or 2-Deoxy-D-glucose) and place back in the incubator for 2h 0m 0s .

Make up the Wash Buffer using the 10X Wash Buffer Concentrate and dilute 1:10 with deionised water.

Once the plate has finished incubating, move over the 500µL supernatant into a new sterile 24-well plate and add DPP-IV inhibitor immediately (1:100).to be carried out in a laminar flood hood. The cells may be stored at -80°C for further analysis at a later date.

In each well of the ELISA plate, add 300µL of diluted Wash Buffer (or in two intervals of 150µL). Incubate at Room temperature for 0h 5m 0s. Decant excess buffer and blot with absorbent towels.

Add 200µL Assay Buffer to NSB (non-specific binding) wells A1, A2.

Add 100µL Assay Buffer to the remaining wells you wish to load your samples in.

Add 100µL standards in ascending order to wells - standards come preprepared.

Then load 100µL of QC1 and QC2 to the plate in seperate wells.

Add 100µL of your samples from the 24-well plate in the remaining wells (already containing 100µL of the Assay Buffer). Plate layout should resemble the below.

For good mixing, lightly agitate the plate.

Cover the plate with plate sealer. Incubate (20 to 24 hours) at 4°C .

All reagents should be warmed to Room temperature before proceeding.

Decant liquid from plate and tap out excess fluid on absorbent towels.

Wash the plate 5 times with 300µL pre-diluted Wash Buffer per well with 0h 5m 0s incubation at Room temperature in Wash Buffer with the fourth wash. Tap out excess buffer on absorbent towels after the fifth wash.

Immediately add 200µL Detection Conjugate (is ready to use) in each well. Incubate 2h 0m 0s at Room temperature then decant.

Whilst the plate is incubating dilute the Substrate. .

Hydrate in 1mL deionized water just before use.

Use at 1:200 dilution in Substrate Diluent (e.g. 100µL hydrated substrate in 20mL substrate diluent). Dilute fresh each time just before use. 

Wash the wells 3 times with 300µL diluted Wash Buffer . Tap out excess buffer on absorbent towels.

Add 200µL diluted Substrate in each well.

Measure fluorescence on a plate reader at an excitation/emission wavelength of 360/460 every 0h 5m 0s for a minimum of 0h 20m 0s .

If sufficient fluorochrome has been generated, add 50µL Stop Solution (mix thoroughly to ensure no precipitate remains) to each well in the same order as the Substrate was added. Incubate 0h 5m 0s at Room temperature in the dark to arrest phosphatase activity.

Read plate on a fluorescence plate reader with an excitation/emission wavelength of 360/460.