TITLE: Mouse genetic models to manipulate enterochromaffin cell activity - Murine Organoid ELISA
AUTHORS: Koki Tohara

[DESCRIPTION]
This protocol describes how we maintain murine intestinal organoids and perform the enzyme-linked immunosorbent assay (ELISA) for serotonin (5-HT).

[STEPS]
SECTION: Preparation or basal media
1. Add 5 mL of GlutaMAX, 1 M HEPES, and Penicillin-Streptomycin to 500 mL of Advanced DMEM/F-12 and filter through 0.22 um. This basal media can last up to1 month.
SECTION: Maintenance of intestinal organoids
31. Put an aliquot of matrigel in fridge at 4°C. Put a 24-well plate to a 37°C incubator.
SECTION: Maintenance of intestinal organoids
32. Put desired volume of complete media at 37°C
SECTION: Maintenance of intestinal organoids
33. Aspirate media from wells
SECTION: Maintenance of intestinal organoids
34. Add 500 µL cold basal media to a well and let it sit for about 15 seconds
SECTION: Maintenance of intestinal organoids
35. Pipette up and down to dissolve matrigel, collect organoids, and move to a 15 mL falcon tube
SECTION: Maintenance of intestinal organoids
36. Add 500 µL cold basal media to a well, collect residual organoids, and move to the same 15 mL falcon tube
SECTION: Maintenance of intestinal organoids
37. Place a 200 µL tip inside a 1000 µL tip and pipette up and down ~25 times to break up organoids
SECTION: Maintenance of intestinal organoids
38. Add 10 mL cold basal media to the falcon tube and mix well
SECTION: Maintenance of intestinal organoids
39. Centrifuge at 200 g for 3 min at 4°C
SECTION: Maintenance of intestinal organoids
40. Aspirate the supernatant (be careful not to aspirate the organoid pellet)
SECTION: Maintenance of intestinal organoids
41. Add 10 mL cold basal media to the falcon tube and mix well
SECTION: Maintenance of intestinal organoids
42. Centrifuge at 200 g for 3 min at 4°C
SECTION: Maintenance of intestinal organoids
43. Aspirate the supernatant (be careful not to aspirate the organoid pellet)
SECTION: Maintenance of intestinal organoids
44. Add 200 µL of ice-cold matrigel to the falcon tube and mix the organoids well
SECTION: Maintenance of intestinal organoids
45. Pipette 50 µL of the matrigel/organoid suspension into the center of each of four wells of a prewarmed 24-well plate to form domes in the center of each well
SECTION: Maintenance of intestinal organoids
46. Place the lid on the culture plate and quickly turn the plate upside down
SECTION: Maintenance of intestinal organoids
47. Incubate at 37°C for 10 minutes to set the matrigel
SECTION: Maintenance of intestinal organoids
48. Turn the plate back upside so that you can apply media to wells. Gently add 500 µL complete media to each well
SECTION: Maintenance of intestinal organoids
49. Add basal media to surrounding wells to prevent the wells from drying
SECTION: Maintenance of intestinal organoids
50. Exchange the media every 3-4 days or whenever the media turns yellow. 
SECTION: Maintenance of intestinal organoids
51. Split again in 6-8 days.
SECTION: Preparation of organoids for ELISA
52. Organoids are grown for 4-6 days
SECTION: Preparation of organoids for ELISA
53. Aspirate the supernatant (be careful not to aspirate the organoid pellet)
SECTION: Preparation of organoids for ELISA
54. Add 500 µL of cold DPBS to a well and let it sit for about 15 seconds
SECTION: Preparation of organoids for ELISA
55. Gently pipette up and down ~10 times with P-1000 to remove matrigel (but not to break the organoids up) and move to a 15 mL falcon tube.
SECTION: Preparation of organoids for ELISA
56. Add 500 µL of cold DPBS to the tube
SECTION: Preparation of organoids for ELISA
57. Put the tube on ice for 5 min 
SECTION: Preparation of organoids for ELISA
58. Gently pipette up and down ~10 times with P-1000
SECTION: Preparation of organoids for ELISA
59. Add 10 mL cold DPBS and spin down at 200 g for 3 min at 4°C
SECTION: Preparation of organoids for ELISA
60. Aspirate the supernatant as much as possible
SECTION: Preparation of organoids for ELISA
61. Add 1 mL cold DPBS and gently pipette up and down ~10 times with P-1000
SECTION: Preparation of organoids for ELISA
62. Add 10 mL cold DPBS and spin down at 200 g for 3 min at 4°C
SECTION: Preparation of organoids for ELISA
63. Aspirate the supernatant as much as possible
SECTION: Preparation of organoids for ELISA
64. Repeat the step #33-35 twice to completely wash the matrigel out
SECTION: Preparation of organoids for ELISA
65. Add 100 µL DPBS + 0.1% BSA and move organoids to an eppendolf tube (it is important to add 0.1% BSA to prevent organoids from sticking to tubes)
SECTION: Preparation of organoids for ELISA
66. Remove the supernatant and add 100 µL Ringer's (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM D-glucose, and 10 mM HEPES-Na [pH 7.4])) buffer for ELISA assay (do not lyse the organoids as you are measuring released serotonin).
SECTION: 5-HT ELISA
67. ELISA is performed according to the manufacturer's protocol (https://www.ldn.de/wp-content/uploads/IFU-BA-E-5900R-V15.0_wz.pdf).
SECTION: Mouse organoid prep
3. Organoids were generated from male Tac1-Cre(+/-);Pet1Flp(+/-);RC::FL-hM3Dq(+/-) or Tac1-Cre(+/-);Pet1Flp(+/-);RC::OFTox(+/-) animals (5-8 weeks old).
SECTION: Preparation of complete organoid culture media
2. Add 800 µL of B-27, Recombinant Murine Noggin (final conc. 100 ng/mL), Mouse EGF Recombinant Protein (final conc. 500 ng/mL), N-Acetyl-L-cysteine (final conc. 0.5 mM),  and 4 mL of R-spondin 2 to 40 mL of basal media. This complete media can last up to 2 weeks.
SECTION: Mouse organoid prep
4. Euthanize the animal under CO2 and spray belly with 70% EtOH.
SECTION: Mouse organoid prep
6. Filet open the intestine along the mesentery.
SECTION: Mouse organoid prep
5. Isolate the whole small intestine and divide the tissue into three pieces. Use the middle piece (jejunum) for organoid generation.
SECTION: Mouse organoid prep
7. Scrape off the villi using a glass coverslip. Scrape well so that all the villi come off. 
SECTION: Mouse organoid prep
8. Cut the intestine into 2-4 mm pieces and move to a 50 mL falcon tube.
SECTION: Mouse organoid prep
9. Add 10 mL ice-cold DPBS. Pipette up and down with 10 mL pipettes.
SECTION: Mouse organoid prep
10. Wait for a couple of minutes until the tissue settles down and discard the supernatant.
SECTION: Mouse organoid prep
11. Repeat the step 9-10 until the supernatant becomes clear (usually 4-5 times).
SECTION: Mouse organoid prep
12. Add 30 mL of ice-cold DPBS + 2 mM EDTA and rock the tube at 4°C for 30 minutes.
SECTION: Mouse organoid prep
14. Wait for a couple of minutes until the tissue settles down and discard the supernatant.
SECTION: Mouse organoid prep
16. Wait for a couple of minutes until the tissue settles down and move the supernatant to falcon tube #1 prepared at step #13.
SECTION: Mouse organoid prep
15. Add 10 mL ice-cold DPBS. Pipette up and down with 10 mL pipettes.
SECTION: Mouse organoid prep
13. While waiting, prepare 6x 50 mL falcon tubes and label them No.1-6.
SECTION: Mouse organoid prep
17. Repeat the step 15-16 five times. Note that the supernatant should be put into separated tubes prepared at step #13 (tube #2-6).
SECTION: Mouse organoid prep
18. Place 20 µL of each supernatant fraction on a glass slide and check under a microscope.
SECTION: Mouse organoid prep
19. Identify fractions that contain crypts.
SECTION: Mouse organoid prep
20. Combine crypts-containing fractions and filter through a 70 µm strainer.
SECTION: Mouse organoid prep
21. Spin down at 900 g for 5 minutes and discard the supernatant. 
SECTION: Mouse organoid prep
22. Resuspend pellet with 10 mL ice-cold basal media and spin down at 900 g for 5 minutes.
SECTION: Mouse organoid prep
23. Carefully remove all the supernatant.
SECTION: Mouse organoid prep
24. Repeat the step #22 and #23.
SECTION: Mouse organoid prep
25. Resuspend the pellet into 400 µL ice-cold matrigel.
SECTION: Mouse organoid prep
26. Pipette 50 µL of the matrigel/organoid suspension into the center of each of four wells of a prewarmed 24-well plate to form domes in the center of each well.
SECTION: Mouse organoid prep
27. Place the lid on the culture plate and quickly turn the plate upside down.
SECTION: Mouse organoid prep
28. Incubate at 37°C for 10 minutes to set the matrigel.
SECTION: Mouse organoid prep
29. Turn the plate back upside so that you can apply media to wells. Gently add 500 µL complete media to each well. 
SECTION: Mouse organoid prep
30. Add basal media to surrounding wells to prevent the wells from drying.