1. Identify mice to be used. This protocol is designed for 4 mice to be simultaneously processed with each mouse serving as individual biological samples. Note: 4 separate samples are the maximum recommended amount for one person.
2. Prepare the following solutions (can be done the night before):
Dissociation solution = 8 x 20 mL (in 50 mL conical tubes) chilled dissociation solution. Make stock = 160 mL PBS + 160 µL of 1000x Rock Inhibitor (Final concentration is 10 µM) + 800 µL of 0.4 M EDTA (Final concentration is 2mM). Aliquot 20 mL each into 8 conical tubes (Dissociation #1 and #2 for each sample; recommended labels ‘Mouse #1 Dissociation #1’ = ‘M#1 Dis #1’ and ‘Mouse #1 Dissociation #2’ = ‘M#1 Dis #2’), keep chilled at 4°C if overnight, and on ice for immediate use.
FACS Buffer (40 mL total) = 38.8 mL PBS + 1.2 mL FBS (Final concentration is 3%) + 40 µL of 1000x Rock Inhibitor (Final concentration is 10 µM).
3. Label 50 mL conical tubes – 4 tubes per mouse. Recommended label for Mouse #1: M#1 – 100f #1; M#1 – 100f #2; M#1 – 40f #1; M#1 – 40f #2 (100f = 100 µm filter; 40f = 40 µm filter).
4. Label 4 petri dishes with mouse ID/#, pour half full with cold PBS, keep on ice.
5. Sacrifice mice and immediate proceed with dissection.
6. Pin mouse on dissection tray, spray abdomen with 70% ethanol, and make incision to open peritoneal cavity. Pin skin back as needed.
7. Gently scoop out the small intestine to the left side. Cut through the pelvis just to the right of the rectum, then find the rectum and cut at the end near the junction with the skin. Avoid cutting blood vessels.
8. Cut the small intestine where it meets the cecum. Gently tug the top of the colon and if cut correctly at the rectum, it will slowly pull free with minimal mesentery tissue.
9. Place the colon in the chilled PBS in the petri dish and keep on ice. Proceed with dissecting remaining mice.
10. Linearize each section of colon – keeping it in PBS on ice: 1) Snip off the tip of the cecum pouch. 2) Fill the 10 mL syringe attached to a popper needle with cold PBS and gently flush out the cecum to remove fecal matter. 3) Refill the syringe as needed and continue to expel PBS while threading the popper needle gently through the colon. Expel all remaining PBS once entire colon is threaded on the popper needle. 4) Prop the plunger end of the syringe with the popper needle/tissue attached in a firm and stable place (for example wedged against the edge of an ice bucket) with popper needle facing you. Place one point of dissecting scissors in the opening of the popper needle – the syringe and popper needle should stay firmly in place while you hold the scissors. With your free hand, use forceps to pull the intestine off the popper needle, towards the scissors, cutting and linearizing as you pull the intestine towards you.
11. Keep linearized colon in PBS, while proceeding with other colons.
12. Rinse colon by swirling in clean PBS until a majority of fecal material is removed.
13. Place clean linearized colons in dissociation solution #1 (as described in preparation step 2) and place at a slow rotation (If using recommended Fisherbrand Multi-Purpose Tube Rotator, use speed setting of 8 rpm with a horizontal axis of rotation ensuring solution and intestines are rotated from the top of the conical tube to the bottom). Dissociate for 30 min at 4 °C.
14. Remove colon tissue from dissociation solution #1. Cut tissue into small ~3-5 mm pieces, using forceps to dangle the tissue above dissociation tube #2 with edge of tissue resting on tube rim to pull taut and cutting with scissors. Make sure all pieces are immersed in the dissociation solution #2. Place back on rotator at 4 °C for an additional 30 min rotation.
15. Turn on centrifuge (swinging bucket preferred) during this period to allow time to chill to 4 °C. Adjust settings: 500 xg, 5 min, 4 °C, decrease deceleration speed to low setting.
16. After the 30 min dissociation step is completed (1 hr in total), collect conical tubes and shake for 3 minutes very aggressively and rapidly (up and down motion). Solution should quickly become cloudy with an observable abundance of floating cells.
17. Pour suspension through 100 µm filter into new conical tube (100 µm filter #1 tube – example label M#1 – 100f #1). Rinse filter with cold PBS (final volume of 40-50 mL). Collect tissue chunks trapped in the filter and place back in the dissociation tube. Re-use this filter for the next 100 µm filter for this sample (move the filter to the conical tube labeled 100 µm filter #2).
18. Add ~20 mL of PBS to the dissociation tube that contains tissue chunks and store on ice. It is important to not allow the tissue chunks to get dry.
19. Repeat steps 17-18 for all samples and then immediately spin down all 100 µm filter #1 tubes at 500 xg, 5 min, 4 °C, with decreased deceleration speed. NOTE: All 4 collection tubes will need to be processed rapidly in sequence so that centrifugation steps are done together. The best rates of cell survival depend on minimizing the time cell suspensions are sitting and ensuring that when they are sitting it is always on ice.
20. Manually shake tissue in the dissociation solution tubes (now with 20 mL PBS) again for 3 minutes rapidly.
21. Filter through 100 µm filter into a new tube (100 µm filter #2 tube). Rinse with PBS for a final volume of ~40-50 mL. Tissue trapped in filter can be saved (as back-up) or discarded. Spin down 100 µm filter #2 tube (and collect 100 µm filter #1 tubes from centrifuge).
22. Gently pour off supernatant from the 100 µm filter #1 tube and resuspend in 1 mL of FACS buffer. Mix well with a pipet to achieve a homogenous suspension. Filter the suspension through a 40 µm filter into a new tube (40 µm filter #1 tube). Rinse the filter with PBS filling to 50 mL. Repeat for all samples and save the 40 µm filters for the next step.
23. Repeat step #22 using the suspension from the second shake (100 µm filter #2 tubes) and filter into 40 µm filter #2 tube using the same 40 µm filter saved from step #22 for each sample.
24. Centrifuge the suspension in both 40 µm filter tubes (#1 and #2) at 500 xg, 5 min, 4 °C, with a decreased deceleration speed to protect cell viability.
25. During the centrifugation step, begin a prep of DNAse in a 1.5 mL Eppendorf tube = 20 µL of 10x buffer (contains MgCl) + 40 µL DNAse I + 140 µL Water.
26. Gently pour off supernatant from the 40 µm filter tubes and resuspend each cell pellet in ~1 mL of FACS buffer, making sure to mix and suspend the cells very well. Transfer the suspension into 1.5 mL Eppendorf tubes (if you end up with a larger volume, it is okay to use additional tubes).
Preparation for FACS
27. Centrifuge at 1,000 xg, 5-10 min, 4 °C, with a soft stop setting on to maintain cell viability. As with all centrifuge steps, repeat if pellet is not good. NOTE: The initial centrifugation, when cell suspensions are well mixed, usually needs a full 10 min, subsequent centrifugations require only 5 minutes.
28. Carefully remove and discard supernatant from all tubes using a pipet. Dispense 500 µL of FACS buffer to each tube – if more than one tube was collected per sample, merge the contents of these into one tube with 500 µL FACS buffer total. Add 50 µL of DNAse and mix. Mechanically mix the DNAse and cell suspension up and down 5-10 times with a P-1000 pipet. Incubate at room temperature for 5 min.
29. Collect the cells by centrifugation, and then remove supernatant carefully. Resuspend cell pellets in a total of ~500 µL of FACS buffer.
30. Add antibodies to the cell suspensions as listed below:
Amount per tube (or recipe for master mix below)
CD117 (cKit) – APC-Cy7 [1:100] 5 µL
CD326 (Epcam)– eFluor450 [1:100] 5 µL
CD44 – PerCP-Cy5.5 [1:100] 5 µL
CD24 – PECy7 [1:200] 2.5 µL
CD31 – BV510 [1:200] 2.5 µL
CD45 – BV510 [1:200] 2.5 µL
Antibody mastermix (for 4 mice/4 tubes) (22.5 µL per tube)
20 µL each of cKit, Epcam and CD44
10 µL each of CD24, CD31, and CD45
NOTE: Add single channel antibody controls as needed to establish compensations and sorting gates. Once these gates are established the protocol is very consistent and these controls are not needed for every sort.
31. Vortex briefly and then incubate for 30 min at 4 °C in the dark.
32. Collect cells by centrifugation (1,000 xg, 5-10 min, 4 °C, with a soft stop setting) and carefully remove supernatant. Add 1 mL fresh FACS buffer, resuspend, and centrifuge again. Remove supernatant (Wash step).
33. Resuspend each suspension thoroughly in ~500 µL - 1mL fresh FACS buffer into 5 mL round bottom tube for sorting (label tube as ‘To Sort’). NOTE: It is recommended to start with a lower resuspension volume for a more concentrated cell suspension enabling faster sorting. Adjust total volume depending on mouse number/condition and cell number since the yield and recommended resuspension volume depends on the quality of the prep.
34. Add Live/Dead Aqua dye (maintain 1 µL Live/Dead per 1 mL of cell suspension), wrap in foil and incubate at room temperature for ~2 min. Incubate at least 5-10 minutes before running samples for FACS but keep samples on ice (it is not necessary to incubate for longer). NOTE: The Live/Dead Aqua dye is only good for 2 weeks once reconstituted.
35. Store all samples on ice, protected from light, until time to sort.
36. If only analysis is being performed, no preparation for sorting is needed. If sorting is occurring, prepare tubes/reagents for collection.
For sorting followed by RNA isolation we recommend sorting directly into TRIzol – a step that preserves RNA integrity. Collection tubes can be FACS tubes or Eppendorf tubes.
For sorting followed by mass spectrometry we changed the sorting machine sheath fluid to 100 mM ammonium bicarbonate and sorted directly into 50 µL of 100 mM ammonium bicarbonate – a step that preserves protein integrity and prevents salt contaminants. We recommend sorting into PCR tubes (that can be fitted inside of Eppendorf tubes) – although this depends on the set-up of downstream mass spectrometry equipment.
Regardless of downstream applications store collection tubes on ice/chilled prior to, during, and after sorting.
37. Populations to collect (live cells):
(CD45-, CD31-, CD326+, CD44highest, CD24-, CD117-)
Absorptive Progenitor [AbsPro]
(CD45-, CD31-, CD326+, CD44med/+, CD24-, CD117-)
Secretory Progenitor + Deep Crypt Secretory + Goblet(minor) [SecPDG]
(CD45-, CD31-, CD326+, CD44 high, CD24med, CD117med)
(CD45-, CD31-, CD326+, CD24+, CD117+)
(CD45-, CD31-, CD326+, CD44-/low, CD24-)
Enteroendocrine Cells [EEC]
(CD45-, CD31-, CD326+, CD44-/low, CD24+)
38. Follow the gating schema provided in Figure 4.
39. Recommended sorting parameters: (1) BD FACS Aria Fusion using a 100 µm nozzle (20 PSI). (2) Flow rate of 2.0 with a maximum threshold of 5,000 events/sec. (3) Keep sample chamber and collection tubes at 4 °C.