Isolation and analyses of lamina propria lymphocytes from mouse intestines

Summary Investigating intestinal immune responses is critical to understanding local and systemic immunity. However, obtaining resident intestinal immune cells with high cell viability can be challenging. Here, we provide an optimized protocol to isolate lamina propria lymphocytes from the small and large intestines, including lymphocyte activation for cytokine expression analysis and techniques for surface and intracellular antibody staining and flow cytometry. This protocol can be used for isolating and analyzing tissue-resident immune cells from other tissues with specified modifications. For complete details on the use and execution of this protocol, please refer to Kim et al. (2022).


SUMMARY
Investigating intestinal immune responses is critical to understanding local and systemic immunity. However, obtaining resident intestinal immune cells with high cell viability can be challenging. Here, we provide an optimized protocol to isolate lamina propria lymphocytes from the small and large intestines, including lymphocyte activation for cytokine expression analysis and techniques for surface and intracellular antibody staining and flow cytometry. This protocol can be used for isolating and analyzing tissue-resident immune cells from other tissues with specified modifications. For complete details on the use and execution of this protocol, please refer to Kim et al. (2022).

BEFORE YOU BEGIN
Institutional permission for animal experiment All experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee of Harvard University, Boston, USA.
The protocol below describes the specific steps for isolating and analyzing intestinal lamina propria lymphoid cells.
Alternatives: This protocol is optimized for intestinal lamina propria immune cells. However, we have also used this protocol for isolating and analyzing tissue-resident immune cells from other tissues (e.g., placenta, liver, brain, meninges), with slight modifications. For example, if working with non-mucosal tissues, EDTA and DTT digestion steps can be skipped and Liberase concentration can be adjusted to 50 mg/mL.
Gut epithelial cells and intraepithelial lymphocytes can also be harvested from this protocol; see note after step 10 for more details.

Mice
The composition of immune cells in the intestinal lamina propria is greatly affected by the gut microbiota (Round and Mazmanian, 2009), as well as sex, age, and strain (Elderman et al., 2018;Man et al., 2014). Therefore, the use of vivarium-, age-, sex-, strain-matched control and experimental groups is required. Animals were purchased from the same barrier and vendor for each experiment. All 1. Prepare the necessary buffers and media before starting the experiment. Recipes and storage conditions for the buffers and media can be found in the materials and equipment section.

Antibody panel preparation
Timing: 0.25-0.5 h 2. Prepare two flow cytometry antibody panels for surface marker proteins (Table 1) and intracellular cytokines/transcription factors (Table 2). Antibodies need to be prepared immediately before the staining step as a master mixture. Detailed staining panel information and dilution factors can be found in the materials and equipment section. HBSS will be used for surface marker proteins antibodies mixture, and 13 permeabilization buffer will be used for intracellular cytokines/transcription factors master mixture.
Note: We used the same fluorophore for CD8a and CD19 because we can differentiate CD8 T cells by gating on TCRb, CD3e-positive and CD8a (BV605), and B cells on TCRb, CD3e-negative, and CD19-positive (BV605). However, different fluorophores can be chosen for either CD8a or CD19 if it is possible to add more fluorophores.

MATERIALS AND EQUIPMENT
Note: Reconstitute the entire vial and aliquot the Liberase stock into 1.5 mL microcentrifuge tubes (1 mL/tube). Gently agitate the vial at 4 C until the enzyme is completely dissolved ($30 min). The stock can be stored at À20 to À80 C. Note: Reconstitute the entire vial and aliquot the DNase I stock into 1.5 mL microcentrifuge tubes (1 mL/tube). Gently agitate the vial at 4 C until the enzyme is completely dissolved ($30 min). Do not vortex to dissolve. The stock can be stored at À20 to À80 C.
Note: Prepare and use on the day of experiment. Prewarm HBSS at 37 C before use.
Note: Prepare and use on the day of experiment. Prewarm RPMI-1640 at 37 C before use.
Note: Prepare and use on the day of experiment. Equilibrate to 20 C-22 C before use. The addition of HBSS to Percoll is required to make Percoll isotonic with physiological conditions and maintain osmotic pressure in cells. Note: Can be prepared in advance and stored at 4 C for at least 4 months. Filtration through a 0.2 um vacuum filter is recommended.
Note: Can be prepared in advance and stored at 4 C for up to 1 month.
Note: Prepare and use on the day of experiment. Prewarm at 37 C before use.
Note: Freshly prepare before use. Note: Freshly prepare before use.

Reagent
Note: Freshly prepare before use.
Note: Freshly prepare before use.
Note: Freshly prepare before use.
Note: Freshly prepare before use.

STEP-BY-STEP METHOD DETAILS
Intestinal tissue collection  Figure 1A). c. Find the terminal colon and cut it free with scissors. Then, gently pull out the intestines. (Figure 1B). d. Take the desired part of the intestines and place in a multi-well-plate with 5 mL of HBSS (w/o Ca 2+ , Mg 2+ ) on ice, while other samples are being processed ( Figure 1C).
CRITICAL: Keep all samples submerged in HBSS and on ice while processing other samples, as dried tissues will have reduced viable cell yield.
Optional: Addition of 2% FBS into HBSS may improve cell viability.

Trimming the tissue
Timing: 5 min/ sample These steps prepare each sample for subsequent dissociation by removing tissues that can interfere with lymphocyte extraction and analysis.
3. Trim the intestine samples (Methods video S1). a. Transfer the intestines onto ice-cold HBSS soaked paper towels and trim the surrounding fat tissues ( Figure 1D) and Peyer's patches ( Figure 1E). b. Remove the intestinal content thoroughly by gently pushing it from one end of the gut and out the other end ( Figure 1F). Alternatively, a syringe can be used to inject HBSS inside of the intestines to remove the intestinal contents. c. Slide in angled forceps into the intestines and cut longitudinally ( Figure 1G). d. Wash the sample twice by placing it into ice-cold HBSS and clean off mucus by gently scrubbing the tissue on the wet paper towel ( Figure 1H). e. Transfer the tissue into a new multi-well plate with 5 mL HBSS on ice while other samples are being processed ( Figure 1I).
CRITICAL: Ensure all Peyer's patches are removed from the gut, as these may greatly impact immune cell composition results. Try to minimize the time that samples are off ice to keep cells alive. Incomplete removal of mucus or tissues drying out can also reduce the yield.

Removing epithelial cells and intraepithelial lymphocytes
Timing: 0.5 h These steps dissociate unwanted cell populations from the intestinal tissues, including epithelial cells and intraepithelial lymphocytes, and leave primarily cells from the lamina propria.  (Figure 2A). 6. Incubate the tissues in a shaker at 37 C, 250 rpm for 15 min ( Figure 2B). a. Place the tubes at a 45 angle for better shaking. 7. Vortex each sample vigorously at maximum speed ($3,000 rpm) for 10 s ( Figure 2C). 8. Prepare 2 Petri dishes with 20 mL ice-cold RPMI-1640 media. 9. Take the tissue out of the EDTA-DTT buffer and remove residual solution by scrubbing the tissue on a clean paper towel ( Figure 2D). 10. Rinse the tissues with ice-cold RPMI-1640 media ( Figure 2E), then scrub on a clean paper towel.
Repeat this step to thoroughly remove dissociated cells.
Note: If epithelial cells or intraepithelial lymphocytes are the desired population for analysis, EDTA (for intestinal epithelial cells) and DTT (for intraepithelial lymphocytes) buffers can be treated separately and the supernatant saved after each reaction.

Isolation of lymphocytes from lamina propria
Timing: 2-2.5 h

OPEN ACCESS
These steps will generate a single cell suspension of the lamina propria cells, then isolate lymphocytes via a Percoll density gradient.
11. Prepare 5 mL digestion media per sample. a. Prewarm RPMI-1640 media at 37 C. c. Prepare a 50 mL conical tube per sample. d. Aliquot 5 mL of digestion buffer to each conical tube.
12. Transfer the tissues to the digestion buffer in the prepared conical tubes ( Figure 2F). 13. Incubate the tissues in a shaker at 37 C, 250 rpm for 40 min to 1 h. a. Place the tubes at a 45 angle for better shaking. b. Vigorous shaking is recommended every 15 min for more efficient digestion.
Note: Optimal digestion time can be varied depending on the tissue type, size, and diseaseinduced conditions. Usually, 35 min incubation for small intestines and 50 min for large intestines are optimal for efficient digestion while keeping high cell viability. For colitis-induced colons, longer incubation is recommended. See troubleshooting 1.
14. Prepare new 50 mL conical tubes with 100 mm cell strainers on the top. 15. Transfer the digested tissues through the strainers into the new tubes ( Figure 2G). a. Vortex vigorously after incubation for 10 s. b. Pour the digested cell suspensions through the cell strainer. Small non-dissociated pieces of tissues can be discarded. c. Add 15 mL ice-cold HBSS to the original 50 mL conical tube to rinse out residual cells and pour through the cell strainer. 16. Centrifuge the samples at 450 3 g for 10 min at 4 C. 17. Carefully remove the supernatant by vacuum suction or pouring out while taking care not to disturb the cell pellet. 18. Prepare 80% Percoll and 40% Percoll solutions, then prepare 40% Percoll tubes per sample.
a. Prepare a 15 mL conical tube for each sample. b. Aliquot 4 mL of 40% Percoll solution to each conical tube. 19. Resuspend the cell pellets with 1 mL of 40% Percoll solution and transfer to the prepared 15 mL conical tube with 4 mL of 40% Percoll solution ( Figure 2H and Methods video S2). 20. Add 2.5 mL of 80% Percoll solution to the bottom of the 15 mL tubes ( Figure 2I and Methods video S2). a. Place a glass Pasteur pipette into each tube with the 40% Percoll cell suspension, making sure the pipette reaches the bottom of the tube. b. Slowly apply 2.5 mL of 80% Percoll solution through the glass Pasteur pipette to the bottom of the tube. c. Wait until the 80% Percoll solution is completely added before removing the glass Pasteur pipettes to leave the 40/80% Percoll interface intact. 21. Gently move the samples to the centrifuge, while not disturbing the 40/80% Percoll interface ( Figure 2J). 22. Centrifuge the samples at 860 3 g for 20 min at 21 C with the lowest acceleration speed (acceleration 0 or 1) and no brake (deceleration 0).
CRITICAL: Lowering acceleration and deceleration speed help create a clearer interface to identify lymphocytes. Centrifugation at 21 C is critical, as the density of Percoll changes at different temperatures.
23. Prepare a new 15 mL conical tube per sample and add 10 mL of HBSS. 24. Take the cells located at the interface of 40/80% Percoll solution ( Figure 2K and Methods video S3). Troubleshooting 2.

OPEN ACCESS
a. Remove the upper 40% Percoll solution from the top until $4 mL of total solution remain (Figure 2L). b. Use a transfer pipette to collect the cells (lymphocytes) at the interface ( Figure 2M). c. Transfer the lymphocytes to the newly prepared 15 mL tube in step 23. 25. Thoroughly mix by inverting and centrifuge at 450 3 g for 10 min at 4 C. 26. Discard supernatant. 27. Add 10 mL of HBSS to the pellet and centrifuge at 450 3 g for 10 min at 4 C to wash the pellet.
CRITICAL: Complete Percoll removal is critical for cell viability, particularly in cases where cell stimulation is required. A second wash with HBSS after step 28 is optional to fully remove Percoll from the samples.

Timing: 2.5-3 h
If lymphocyte stimulation is required, these steps will activate the isolated lymphocytes for cytokine expression analyses.
29. Prewarm the 23 T cell stimulation media and aliquot 100 mL into a 96-well round bottom plate for each sample, plus one well for the unstained control. 30. Resuspend each sample cell pellet from step 28 with base T cell culture media.
Note: The volume of media for the resuspension can be adjusted based on the size of the cell pellet. 1 3 10 6 to 2 3 10 6 cells are recommended for the stimulation. Count the cell number using a Burker chamber or automated cell counter with Trypan Blue staining for live/dead cell discrimination for the representative sample and determine the total volume of the resuspension. e.g., If 3 3 10 6 cells are harvested and 1 3 10 6 are desired for stimulation, resuspend the cell pellets with 300 mL of the base T cell culture media. If the number of cells in a specific cell population needs to be calculated, please remember the resuspension volume in case you need to calculate back the cell numbers after flow cytometry analyses. Troubleshooting 3. 31. Take 100 mL of each sample cell suspension and add it to the corresponding well containing 23 T cell stimulation media. 32. Thoroughly mix by pipetting gently. 33. Take 100 mL of any residual cell suspension and add it to the unstained control well.
Optional: If fluorescence minus one staining (FMO) controls are required (Tung et al., 2007), save 100 mL of any residual cell suspension into additional wells.
34. Incubate the 96-well plate in a 5% CO 2 , 37 C cell culture incubator for 2-3 h. Note: This step can be combined with antibody staining by creating a single master mix with both components. This has demonstrated comparable results to keeping these mixtures separate, but the efficacy may differ depending on antibodies.
46. Add 150 mL/well of HBSS to wash and centrifuge at 450 3 g for 2 min at 4 C. 47. Remove the supernatant by flipping the 96-well plate. 48. Prepare antibody mix 1 as indicated in Table 1 for surface markers staining (Surface markers staining panel). 49. Add 50 mL/well of antibody mixture and resuspend cells gently. 50. Incubate at 4 C for 30 min. The plate should be covered with aluminum foil. 51. Add 150 mL/well of HBSS to wash and centrifuge at 450 3 g for 2 min. 52. Remove the supernatant by flipping the 96-well plate. 53. Repeat steps 51 and 52.
Note: If intracellular staining is unnecessary, skip steps 54-65 and proceed to step 66.

Intracellular cytokines and transcription factors staining for flow cytometry analyses
Timing: 1.5 h These steps will fix and permeabilize the lymphocytes and stain with antibodies for intracellular cytokines and transcription factors. Optional: If cell numbers need to be counted, add counting beads (Cat. 424902, Biolegend) to each well according to the manufacturer's protocol (https://www.biolegend.com/en-ie/ products/precision-count-beads-13279). Cell numbers can be calculated back after flow cytometry runs.

Data collection
66. Collect data with flow cytometry analyzer (LSRII, BD Biosciences) and analyze by using FlowJo software (FlowJo, LLC). Prior to running the samples, appropriate PMT voltage and compensation are required. The usage of compensation beads with the same set of antibodies from each panel is recommended. Compensation control for Fixable live/dead aqua can be prepared with amine-reactive compensation bead kit. Troubleshooting 5 and 6.

EXPECTED OUTCOMES
This protocol outlines the purification of live lymphocyte populations from the mouse lamina propria for analysis of gut mucosal immune cell populations and their perturbations. Using this protocol, we expect to obtain up to 80% of viable intestinal lamina propria lymphocytes, and can define B cells (AmCyan -CD45 + TCRb -CD3e -CD19 + ), non-ab T cells (likely gd T cells, AmCyan -CD45 + TCRb -CD3e + ), CD8 + T cells (AmCyan -CD45 + TCRb + CD3e + CD19 -CD4 -CD8 + ), CD4 + T cells (AmCyan -CD45 + TCRb + CD3e + CD19 -CD8 -CD4 + ), and subsets of CD4 + T cells such as IL-17A-, IFNg-, Th17,and regulatory T cells). With the range of cell types that can be detected, this protocol enables the detailed study of broad intestinal immune cell composition and how different treatments or states affect the differentiation of specific immune cell types as well as overall gut immune tone.
Our protocol can be used to characterize the immune cell compositions of different tissue types, such as the large ( Figure 3A) and small intestines ( Figure 3B) which are immunologically distinct within the same individual. The composition of immune cells in the intestinal lamina propria is also greatly affected by vivarium. For example, in mice that are colonized with segmented filamentous bacteria (SFB), almost 15-20% of all CD4 + T cells in the small intestine (particularly ileum) are IL-17A-producing Th17 cells (Ivanov et al., 2009) (Figure 3B), while in SFB-negative mice this proportion is less than 5%. These observations underscore the importance of including proper control groups during experimental design, but also highlight the sensitivity of this protocol in detecting the impact of small-scale changes.

LIMITATIONS
The antibody panels that we used in this protocol are not sufficient to analyze all lymphocytes population in the intestinal lamina propria, such as ILCs. For analyzing lymphocyte populations other than T cells, antibody panels for surface, intracellular, and transcription factor staining can be modified (Wang et al., 2020). Additionally, as noted earlier, intestinal immune cell composition can be highly sensitive to a variety of factors, including housing conditions and microbial exposures. This may greatly affect results if the desired immune cell populations are over-/under-induced at baseline due to such confounding factors, so it is imperative that experiments are controlled accordingly.

TROUBLESHOOTING Problem 1
Tissues are not completely digested (step 13).

Potential solution
Intestinal tissues from inflammation-induced conditions (e.g., colitis) can be thicker and tougher compared to the homeostatic condition. Increasing the frequency of vigorous shaking during the digestion or 10-15% extension of digestion time can be helpful up to 1 h. Chopping the tissues prior to digestion may also help, but is not required. Excessive mechanical dissociation may lower the cell viability.

Problem 2
Few to no cells are visible at the 40/80% Percoll interface (step 24).

Potential solution
When trying to analyze the lamina propria lymphocytes from a specific location of small intestinal tissue (e.g., duodenum or terminal ileum) in homeostatic condition, cells may not be clearly visible at 40/80% Percoll interface. Even if the interface is faint, we recommend proceeding with the protocol.
If too many fat tissues remain after tissue trimming, or mucus is not completely removed after the EDTA-DTT buffer incubation steps, lymphocytes may become trapped in these tissues which will prevent the pelleting of lymphocytes from the single-cell suspension.

Potential solution
Flow cytometry results can still be reliable with a small number of cells. Even if the cell yield does not reach optimal numbers, cell stimulation can still proceed with the same conditions.
To increase cell yield, make sure to extract as many of the cells at the 40/80% Percoll interface as possible while avoiding disturbing the 80% layer. Thorough removal of fat and mucus during trimming and digestion steps may also help to increase cell counts. Care when pipetting cells and removing supernatant, especially at small volumes, will also raise final cell yields.

Potential solution
After the fixation and permeabilization step, it is expected that the cell pellets become less visible, particularly in the case that the starting numbers of cells were small. To avoid the loss of fixed/permeabilized cells, we typically increase the speed of centrifugation to 860 3 g after cells have been fixed (step 56). We also recommend moving forward with the protocol, as we have found that samples with undetectable cell pellets still often yield meaningful cell counts. Problem 5 Low cell viability (step 66).

Potential solution
Optimal usage of Liberase concentration and digestion time are critical to cell viability. To immediately stop the digestion reaction immediate addition of 10 mL ice-cold HBSS to the digestion buffer is helpful. Complete removal of Percoll before stimulating the cells with stimulation media can be also critical. Residual Percoll can be confirmed under the microscope.
In addition, we have observed that exposure to specific microbes can impede the isolation of viable immune cells from the lamina propria. Finely controlling housing conditions such as maintaining cages independently to limit cross-exposure from handling other mice can improve yield if this is an issue.

Problem 6
Antibody staining of cell markers is ineffective (step 66).

Potential solution
We recommend designing antibody panels such that the brightest antibodies are used for targets with the lowest expression levels. For instance, in our antibody panel, we have used PE and APC for weak targets (RORgt and FoxP3) which we found improved their detection. There are several online tools that may assist in optimizing antibody panel design (https://www.bdbiosciences.com/ content/dam/bdb/marketing-documents/Fluorochrome-Chart-Relative-Brightness.pdf).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jun R. Huh (Jun_Huh@hms.harvard.edu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate or analyze any datasets.

DECLARATION OF INTERESTS
Jun R. Huh is a co-founder of Interon laboratories. He is also a consultant for CJ Bioscience.