Aug 03, 2023
Mouse genetic models to manipulate enterochromaffin cell activity - Murine Organoid ELISA
- 1University of California, San Francisco
Protocol Citation: Koki Tohara 2023. Mouse genetic models to manipulate enterochromaffin cell activity - Murine Organoid ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jz54l1b/v1
Manuscript citation:
Gut Enterochromaffin Cells are Critical Drivers of Visceral Pain and Anxiety (https://www.biorxiv.org/content/10.1101/2022.04.04.486775v1)
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 12, 2022
Last Modified: August 03, 2023
Protocol Integer ID: 71277
Keywords: Intestinal organoid, Serotonin, 5-HT, ELISA
Funders Acknowledgement:
Damon Runyon Cancer Research Foundation
Grant ID: 2387-30
Abstract
This protocol describes how we maintain murine intestinal organoids and perform the enzyme-linked immunosorbent assay (ELISA) for serotonin (5-HT).
Materials
DPBS, no calcium, no magnesium (Thermo Fisher #14190144)
DPBS, no calcium, no magnesium + 2 mM EDTA
Advanced DMEM/F-12 (Thermo Fisher #12634010)
1 M HEPES (Thermo Fisher #15630080)
GlutaMAX™ Supplement (Thermo Fisher #35050061)
Penicillin-Streptomycin (10,000 U/mL) (Thermo Fisher #15140163)
Recombinant Murine Noggin (Peprotech #250-38)
Mouse EGF Recombinant Protein (Thermo Fisher #PMG8041)
N-Acetyl-L-cysteine (Millipore-Sigma #A7250-5G)
B-27™ Supplement (50X), serum-free (Thermo Fisher #17504044)
R-spondin 2 (supernatant from R-spondin expressing HEK293 cells)
Corning® Matrigel® Matrix (Corning #356231)
Bovine Serum Albumin (Millipore-Sigma #A9418)
Serotonin ELISA kit (LDN #BA-E-8900)
24-well plate (Falcon® 24-Well Plate #38021)
Corning™ Falcon™ Cell Strainers 70 µm (Corning #08-771-2)
Ringer's solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM D-glucose, and 10 mM HEPES-Na [pH 7.4]))
Following four mouse lines were used.
RC::FL-hM3Dq (Jackson Labs, Strain #026942)
Tac1Cre (Jackson Labs, strain #021877)
Pet1Flp (gift of Dr. Susan Dymecki)
RC::PFTox (gift of Dr. Susan Dymecki)
Preparation or basal media
Preparation or basal media
10m
10m
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.
Preparation of complete organoid culture media
Preparation of complete organoid culture media
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.
Mouse organoid prep
Mouse organoid prep
10m
10m
Organoids were generated from male Tac1-Cre(+/-);Pet1Flp(+/-);RC::FL-hM3Dq(+/-) or Tac1-Cre(+/-);Pet1Flp(+/-);RC::OFTox(+/-) animals (5-8 weeks old).
Euthanize the animal under CO2 and spray belly with 70% EtOH.
Isolate the whole small intestine and divide the tissue into three pieces. Use the middle piece (jejunum) for organoid generation.
Filet open the intestine along the mesentery.
Scrape off the villi using a glass coverslip. Scrape well so that all the villi come off.
Cut the intestine into 2-4 mm pieces and move to a 50 mL falcon tube.
Add 10 mL ice-cold DPBS. Pipette up and down with 10 mL pipettes.
Wait for a couple of minutes until the tissue settles down and discard the supernatant.
Repeat the step 9-10 until the supernatant becomes clear (usually 4-5 times).
Add 30 mL of ice-cold DPBS + 2 mM EDTA and rock the tube at 4°C for 30 minutes.
While waiting, prepare 6x 50 mL falcon tubes and label them No.1-6.
Wait for a couple of minutes until the tissue settles down and discard the supernatant.
Add 10 mL ice-cold DPBS. Pipette up and down with 10 mL pipettes.
Wait for a couple of minutes until the tissue settles down and move the supernatant to falcon tube #1 prepared at step #13.
Repeat the step 15-16 five times. Note that the supernatant should be put into separated tubes prepared at step #13 (tube #2-6).
Place 20 µL of each supernatant fraction on a glass slide and check under a microscope.
Identify fractions that contain crypts.
Combine crypts-containing fractions and filter through a 70 µm strainer.
Spin down at 900 g for 5 minutes and discard the supernatant.
Resuspend pellet with 10 mL ice-cold basal media and spin down at 900 g for 5 minutes.
Carefully remove all the supernatant.
Repeat the step #22 and #23.
Resuspend the pellet into 400 µL ice-cold matrigel.
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.
Place the lid on the culture plate and quickly turn the plate upside down.
Incubate at 37°C for 10 minutes to set the matrigel.
Turn the plate back upside so that you can apply media to wells. Gently add 500 µL complete media to each well.
Add basal media to surrounding wells to prevent the wells from drying.
Maintenance of intestinal organoids
Maintenance of intestinal organoids
46m
46m
Put an aliquot of matrigel in fridge at 4°C. Put a 24-well plate to a 37°C incubator.
1m
Put desired volume of complete media at 37°C
1m
Aspirate media from wells
2m
Add 500 µL cold basal media to a well and let it sit for about 15 seconds
2m
Pipette up and down to dissolve matrigel, collect organoids, and move to a 15 mL falcon tube
3m
Add 500 µL cold basal media to a well, collect residual organoids, and move to the same 15 mL falcon tube
3m
Place a 200 µL tip inside a 1000 µL tip and pipette up and down ~25 times to break up organoids
3m
Add 10 mL cold basal media to the falcon tube and mix well
1m
Centrifuge at 200 g for 3 min at 4°C
3m
Aspirate the supernatant (be careful not to aspirate the organoid pellet)
2m
Add 10 mL cold basal media to the falcon tube and mix well
1m
Centrifuge at 200 g for 3 min at 4°C
3m
Aspirate the supernatant (be careful not to aspirate the organoid pellet)
1m
Add 200 µL of ice-cold matrigel to the falcon tube and mix the organoids well
2m
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
3m
Place the lid on the culture plate and quickly turn the plate upside down
1m
Incubate at 37°C for 10 minutes to set the matrigel
10m
Turn the plate back upside so that you can apply media to wells. Gently add 500 µL complete media to each well
2m
Add basal media to surrounding wells to prevent the wells from drying
2m
Exchange the media every 3-4 days or whenever the media turns yellow.
Split again in 6-8 days.
Preparation of organoids for ELISA
Preparation of organoids for ELISA
38m
38m
Organoids are grown for 4-6 days
Aspirate the supernatant (be careful not to aspirate the organoid pellet)
2m
Add 500 µL of cold DPBS to a well and let it sit for about 15 seconds
1m
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.
2m
Add 500 µL of cold DPBS to the tube
2m
Put the tube on ice for 5 min
5m
Gently pipette up and down ~10 times with P-1000
2m
Add 10 mL cold DPBS and spin down at 200 g for 3 min at 4°C
3m
Aspirate the supernatant as much as possible
1m
Add 1 mL cold DPBS and gently pipette up and down ~10 times with P-1000
2m
Add 10 mL cold DPBS and spin down at 200 g for 3 min at 4°C
3m
Aspirate the supernatant as much as possible
1m
Repeat the step #33-35 twice to completely wash the matrigel out
12m
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)
1m
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).
1m
5-HT ELISA
5-HT ELISA
ELISA is performed according to the manufacturer's protocol (https://www.ldn.de/wp-content/uploads/IFU-BA-E-5900R-V15.0_wz.pdf).