TITLE: Measurement of GLP-1 release in cell supernatant from Hutu-80 enteroendocrine cells via ELISA 
AUTHORS: r.mezabrovschi, rachel.bates

[DESCRIPTION]
This protocol describes the method of use for the Glucagon-Like Peptide-1 (Active) ELISA Kit
96-Well Plate (Cat. # EGLP-35K) to measure GLP-1 release (pM) from supernatant. The assay should be run in duplicate. 

[BEFORE_START]
All reagents should be warmed to room temperature before proceeding.

[STEPS]
SECTION: Day 2
5. Make up the Wash Buffer using the 10X Wash Buffer Concentrate and dilute 1:10 with deionised water. 


SECTION: Day 2
7. In each well of the ELISA plate, add 300 µL  of diluted Wash Buffer (or in two intervals of 150 µL). Incubate at  Room temperature for 5 min. Decant excess buffer and blot with absorbent towels.

SECTION: Day 2
8. Add 200 µL  Assay Buffer to NSB (non-specific binding) wells A1, A2.

SECTION: Day 2
9. Add 100 µL Assay Buffer to the remaining wells you wish to load your samples in. 

SECTION: Day 2
10. Add 100 µL  standards in ascending order to wells - standards come preprepared. 

SECTION: Day 2
11. Then load 100 µL of QC1 and QC2 to the plate in seperate wells. 

SECTION: Day 2
12. Add 100 µL of your samples from the 24-well plate in the remaining wells (already containing 100 µL  of the Assay Buffer). Plate layout should resemble the below. 

 

SECTION: Day 2
13. For good mixing, lightly agitate the plate. 
SECTION: Day 2
14. Cover the plate with plate sealer. Incubate   (20 to 24 hours) at 4 °C .

SECTION: Day 3
15. All reagents should be warmed to Room temperature  before proceeding.

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

SECTION: Day 3
16. Wash the plate 5 times with 300 µL pre-diluted Wash Buffer per well with 5 min  incubation at Room temperature  in Wash Buffer with the fourth wash. Tap out excess buffer on absorbent towels after the fifth wash.

SECTION: Day 3
17. Immediately add 200 µL Detection Conjugate (is ready to use) in each well. Incubate 120 min at Room temperature  then decant.

SECTION: Day 3
18. Whilst the plate is incubating dilute the Substrate.  . 
SECTION: Day 3
18.1. Hydrate in 1 mL  deionized water just before use. 


SECTION: Day 3
18.2. Use at 1:200 dilution in Substrate Diluent (e.g. 100 µL hydrated substrate in 20 mL  substrate diluent). Dilute fresh each time just before use. 

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

SECTION: Day 3
20. Add 200 µL  diluted Substrate in each well. 

SECTION: Day 3
21. Measure fluorescence on a plate reader at an excitation/emission wavelength of 360/460 every 5 min for a minimum of 20 min . 

 
SECTION: Day 3
22. If sufficient fluorochrome has been generated, add 50 µL  Stop Solution (mix thoroughly to ensure no precipitate remains) to each well in the same order as the Substrate was added. Incubate 5 min  at Room temperature  in the dark to arrest phosphatase activity.

SECTION: Day 3
23. Read plate on a fluorescence plate reader with an excitation/emission wavelength of 360/460. 
SECTION: Day 1
1. 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. 

SECTION: Day 2
2. Check cells are healthy under a microscope. 
SECTION: Day 2
3. In a laminar flow hood aspirate medium and wash with PBS 3 times, adding 500 µL each time. 

SECTION: Day 2
4. Once the final PBS wash is complete add in 500 µL 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 120 min . 

SECTION: Day 2
6. Once the plate has finished incubating, move over the 500 µL 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.