Dissociation and flow cytometric isolation of murine intestinal epithelial cells for multi-omic profiling

Summary Intestinal epithelium is composed of several cell types, which can be dissociated but difficult to maintain high cell viability due to anoikis. Herein, we describe a step-by-step protocol for the isolation of highly viable intestinal epithelial cells using ethylenediaminetetraacetate acid and TrypLE Express, which can subsequently be employed for multi-omic analyses, including single-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Ge et al. (2022).1


SUMMARY
Intestinal epithelium is composed of several cell types, which can be dissociated but difficult to maintain high cell viability due to anoikis. Herein, we describe a step-by-step protocol for the isolation of highly viable intestinal epithelial cells using ethylenediaminetetraacetate acid and TrypLE Express, which can subsequently be employed for multi-omic analyses, including single-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to . 1

BEFORE YOU BEGIN
Our protocol describes in detail the required steps for the isolation and analysis of epithelial cells derived from mouse distal small intestine (ileum). The same protocol can also be used for isolating cecal and colonic epithelial cells. The protocol focuses on the transcriptomic, and metabolomic analysis of ileal epithelial cells at homeostasis, but we have also used it to describe changes in epithelial cell transcriptome and metabolome following intestinal Salmonella Typhimurium infection.
1. Animal studies must be approved by an Institutional Animal Care and Use Committee (IACUC) and performed in accordance with IACUC guidelines. 2. Mouse strain selection will depend on the experiment. We have extensively analyzed 1-12-weekold C57BL/6J mice. 3. Prepare solutions in a biosafety cabinet and store them at 4 C or on ice prior to procedures (15 min; refer to materials and equipment for recipe tables

MATERIALS AND EQUIPMENT
Note: Use magnesium-and calcium-free DPBS.
Alternatives: PBS can be used to replace DPBS in this protocol.
CRITICAL: Do not add sodium azide into FACS buffer as it may reduce cell viability and inhibit cell metabolic activity.

Reagent
Final concentration Amount

Collection of intestinal tissues
Timing: 8 min 1. Euthanize mouse by cervical dislocation or other approved method and expose the abdominal cavity. a. Lay euthanized mouse on its back on a hard surface (e.g., Rodent Surgery Board). b. Lift the skin of the lower abdomen with tweezers and make a V-shape incision with scissors; c. Cut away skin to expose abdominal cavity. 2. Collect ileum tissues.
a. Cut the colon from the anus and gently pull out the intestine from the abdominal cavity. b. Cut the end of the small intestine from the cecum. c. Take the similar length as colon moving proximal from the ileocecal junction (ileum). 3. Remove the fat and mesentery tissues from dissected ileum with tweezers; Flush away the luminal contents using cold DPBS with an animal feeding needle. 4. Using scissors, cut open the ileum longitudinally. Place tissues in 50 mL falcon tube pre-filled with 10 mL cold DPBS and keep the tube on ice until all animals are processed.
CRITICAL: After tissue dissection, perform all procedures as fast as possible and keep all buffers and preparations at 4 C or on ice unless specified otherwise.

Preparation of epithelial single-cell suspension
Timing: 60 min 5. Wash the tissue three times with cold DPBS. a. Remove DPBS by pouring it out. b. Add 10 mL of cold DPBS and shake the tube up and down for 20-30 s. c. Repeat steps 6a and 6b two more times. 6. Transfer tissue to a petri dish and slice into small fragments roughly 2-3 cm in length. 7. Transfer tissue fragments into 15 mL falcon tube pre-filled with 7 mL of cold EDTA buffer and keep the tube on ice. a. Shake the tube by hand (up and down for about 60 cycles; 0.5 s/cycle) every 7-8 min. b. After 30 min of ice incubation, shake the tube vigorously for 2 min. c. Use a tweezer to remove the tissues from the tube. 8. Centrifuge at 500 g and 4 C for 5 min; remove supernatant and add 10 mL of cold DPBS to wash cell pellets. 9. Pellet the cells and add TrypLE Express Enzyme to the tube.

Reagent
a. Spin down the cells at 500 g and 4 C for 5 min. b. Remove the supernatant by pouring. Using pipette tips, remove the residual supernatant. c. Immediately add 2 mL TrypLE Express Enzyme and gently resuspend cell pellets using 1 mL pipette tips. d. Keep at 22 C-25 C for 10 min; During incubation, pipette up and down 10 times with 1 mL pipette tips to gently break cell aggregates every 3-4 min.
Note: Room-temperature (22 C-25 C) incubation is recommended in this step as higher temperature (37 C) may impact cell viability.
10. Fill the tube with 10 mL of cold DPBS and pass through 70 mm cell strainer placed on a 50 mL falcon tube; add another 10 mL of cold DPBS to wash the cell strainer; collect all the flowthrough (single-cell suspension). 11. Centrifuge at 500 g and 4 C for 5 min. Cell pellets are ready for staining. Note: If cells do not pass through cell strainer by gravity, a short centrifugation step can be applied.

Flow cytometry and cell sorting
Timing: (20 min per sample)

Acquire samples on FACS machine and sort cells into post-sort buffer.
Note: For our studies, we used SH800S cell sorter (Sony, Japan) and CytoFlex SRT cell sorter (Beckman Coulter, USA) and analyzed the data using FlowJo software package (Tree Star, USA).
19. Mix cells with AOPI staining solution at 1:1 ratio; Load 20 mL onto slide and count using Auto 2000. Adjust volume to obtain the required cell recovery target.
Alternatives: Cells can also be counted using a hemocytometer.

OPEN ACCESS
Note: The actual number of cells is about 65%-80% of counts recorded by cell sorter.
20. After cell counting, immediately proceed to downstream analysis: a. If cells are used for bulk RNA-seq, spin down the cells at 500 g and 4 C for 5 min and carefully remove supernatant by pipetting; resuspend cell pellets in 350 mL of Buffer RLT Plus supplemented with 1% b-mercaptoethanol.
CRITICAL: Removal of supernatants as much as possible is crucial for ensuring efficient cell lysis. We normally keep less than 20 mL of supernatants in the tube so that the cell pellets are not disturbed. CRITICAL: Wear a mask when handling b-mercaptoethanol.
b. If cells are used for single-cell RNA-seq, immediately proceed to GEM generation and barcoding (https://www.10xgenomics.com/support/single-cell-immune-profiling/documentation/steps/ library-prep/chromium-single-cell-5-reagent-kits-user-guide-v-2-chemistry-dual-index). c. If cells are used for metabolomic analysis, spin down the cells at 500 g and 4 C for 5 min and carefully remove supernatant by pipetting; resuspend cell pellets in1 mL cold DPBS and transfer to 1.5 mL Eppendorf tube for washing. Pellet the cells by centrifugation at 500 g and 4 C for 5 min; completely remove the supernatants.
Pause point: Cells can be snap-frozen in liquid nitrogen or on dry ice and store at À80 C for months until metabolite extraction.

EXPECTED OUTCOMES
This protocol allows for successful isolation of all expected intestinal epithelial cell types. 2 For sample plots of flow cytometry analysis see Figure 1.
Successful dissociation and cell sorting should yield a viability of 75% or greater for optimal downstream application results. The cell number is dependent on the size and health status of the tissue. The ileum of healthy mice (8 weeks old) will typically yield > 200K cells. For single cell sequencing, the cDNA should yield a distinct peak between 400 bp and 10 kb ( Figure 2).

LIMITATIONS
This protocol was only tested on mouse ileum, cecum and colon and may need to be adapted for other mucosal sites.
Red blood cell lysis may lead to a significant reduction in cell viability and/or count.
Insufficient dissociation during TrypLE digestion (e.g., without pipetting up and down) may result in lower cell count.  Potential solution Before adding TrypLE Express, remove leftover supernatants by pipetting as dilution of TrypLE Express reduces its enzymatic activity. During incubation with TrypLE Express, make sure to pipette up and down every 3-4 min to break intestinal crypts. The digestion mixture should be homogeneous with no visible crypts after 10 min incubation.

Problem 2
Loss of cell pellets after TrypLE Express digestion (step 12).

Potential solution
Cell pellet may be loose in 50 mL falcon tube after centrifugation. Always check the cell pellets and gently pour the supernatants out.

Problem 3
High red blood cell number in the single cell resuspension.

Potential solution
Remove mesentery tissues and blood vessels from the intestine as much as possible. Blood removal from anesthetized mouse via heart-puncture could help to reduce red blood cells. Make sure to include anti-mouse TER-119 antibody to exclude red blood cells during cell sorting.

Problem 4
Low yield during cell sorting.

Potential solution
Ensure the antibody dilutions and gating strategy.

Potential solution
Timely processing of tissue after dissection: Complete the tissue processing procedure as soon as possible following tissue collection. Except for TrypLE enzymatic digestion, keep all buffers and samples at 4 C or on ice as indicated. Sort cells right after cell staining and make sure to maintain the sample chamber and collection area at 4 C during cell sorting.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mansour Mohamadzadeh (Zadehm@uthscsa.edu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate new data or code.