Isolation of single crypt cells

Mice were killed at 35 weeks of age, and duodenum and jejunum (10 cm from the stomach)
were harvested and rinsed three times by using a jet of cold 1 phosphatebuffered saline (PBS).
Then, the intestinal tubes were opened longitudinally and the villi were scraped using a
coverslip, followed by washing three times in cold 1 PBS. The intestinal tubes were then cut
into 23 mm pieces and suspended in 1 PBS + 2% fetal bovine serum (FBS). The 23-mm
pieces were extensively washed (1015 times) with 1 PBS + 2% FBS until the supernatant
became clear. Then, the 23-mm pieces were treated with 50 mM EDTA/ 1 PBS for 30 min at
4C on a rocking platform to dissociate the crypts from the intestinal tubes. The dissociated
crypts were passed through a 70-m cell strainer, washed once with 1 PBS, and treated with
TrypLE Express (Life Technologies, Carlsbad, CA, USA) for 30 min at 37C to dissociate cellto-cell attachment. Then, the dissociated cells were passed through a 40-m strainer and
subsequently, through a 20-m strainer. The strained cells were pelleted, resuspended in 1 PBS
+ 2% FBS, and used as single crypt cells.

Epithelium dissociation/FACS
To isolate intestinal crypt cells for fluorescence-activated cell sorting (FACS), small intestine
epithelium was dissociated into single cells essentially as previously described (13) with slight
modifications. For FACS experiments, mouse intestine were flushed with cold PBS, cut open
lengthwise in 10-cm-long pieces, and immersed in PBS/30 mM EDTA/1.5 mM DTT over ice for
20 min. The solution was disposed of, and the tissue was shaken vigorously in fresh
PBS/30mMEDTA for 30 s before being incubated at 37C for 10 min. Intact tissue was
discarded, and dissociated crypts and villi were pelleted at 2,500 revolution/min for 5 min. The
cells were washed twice with cold PBS, resuspended in HBSS/0.3 U/ml dispase at 37C, and
shaken approximately every 2 min for 10 min. Then, (5%) and 100 g DNaseI was added before
the cells were passed through a 70-m filter. Cells were pelleted at 2,500 revolution/min for 5
min and resuspended in 4 ml HBSS with 5% FBS, then passed through a 100-m filter and
combined with an additional 100 g DNaseI.

Cell preparation and SP staining/analysis

Epithelial cells were isolated from the jejunum of mice using our previous published EDTA
method (von Furstenberg et al., 2011). For SP analysis, cells from the preparation were incubated
in SP buffer made with 2% FBS, and 10 mM HEPES (Life Technologies) in HBSS (Life
Technologies) and either Hoechst 33342 (10 g/ml equal to 17 M) (Sigma-Aldrich) for 90 min
at 37 C. Following the incubation, the cells were washed with ice cold HBSS and labeled with
propidium iodide (Sigma-Aldrich) and CD45-FITC (Biolegend) at 0.5 g/10 6 cells for removal of
cells that were dead and/or of hematopoietic origin. For validation purposes, in order to block
vital dye efflux, cells were pre-incubated with 100 M verapamil (Sigma-Aldrich) for 20 min at
37 C and incubated with the above SP solution for 90 min at 37 C with the addition of 100 M
verapamil. To generate the SP fluorescent phenotype, Hoechst 33342 samples were excited using
a UV laser on a MoFlo (Beckman Coulter) fluorescence activated cell sorting (FACS) machine.
Corresponding band-pass filter sets were: MoFlo (blue = 45050 nm, red = 670-30 nm). On each
machine, the SP was defined on the redlo population which was eliminated by verapamil. For
analyses and cell collections, the SP was subdivided into two regions: upper SP (USP) and lower
SP (LSP).

Control

Verapamil

Epithelial isolation and dissociation:

A segment of the proximal intestine representing a 10-cm region from 2 to 12 cm
distal to the pyloric sphincter was used for cell isolation (Fig. 1, n 3 mice). The
intestinal segment was flushed with cold PBS, longitudinally cut open and lightly
rinsed to remove residual luminal contents. Tissue was incubated in PBS containing
30 mmol/l EDTA and 1.5 mmol/l dithiothreitol on ice for 20 min. Intestinal tissue was
transferred to a 15-ml conical tube containing 5 ml of 30 mmol/l EDTA made in PBS,
incubated at 37C for 8 min, then shaken by hand along the tubes long axis.
Shaking frequency and duration were standardized to 2.53 shake cycles per second
for 30 s.
After shaking, the remnant muscle layer was removed and the dissociated cells
were pelleted at 1,000 g, resuspended in PBS 10% FBS, and repelleted. The cells
were then incubated in 10 ml of modified HBSS containing 0.3 U/ml of dispase
(Roche, San Francisco, CA) at 37C for 10 min with intermittent shaking (33.5
cycles per s for 15 s) every 2 min for 15 s to dissociate epithelial sheets to single
cells. After 10 min, a 10-l sample of the cell solution was collected and viewed under
light microscopy to monitor dissociation to single cells. If less than 50% of the tissue
was single cells, the tissue underwent a second round of 30 s shaking and 2-min
incubation at 37C. To enhance cell viability and decrease cell clumping, 1 ml of FBS
and 1,000 U DNase I (Roche) were added to cell suspension, which was sequentially
passed through 70-m then 40-m filters (BD Falcon, Bedford, MA). Cells were
pelleted, then resuspended in 10 ml of HBSS to inactivate the dispase. Cells were
repelleted and resuspended at a concentration of 2 107 cells/ml in IESC media
[Advanced DMEM/F12 (GIBCO, Grand Island, NY) supplemented with 1 N2
(Invitrogen), 1 B27 without vitamin A (Invitrogen), 10 mM HEPES (GIBCO), 10 M
Y27632 (Sigma), 100 g/ml penicillin-streptomycin, and 2 mM L-glutamine]. Cell
viability was assessed by Trypan blue (Invitrogen) staining; a 75% cell viability was
the threshold to proceed forward. An