Preparation of single-cell suspensions

Prepare all buffers and pre-warm at 37C

1. Prepare 12 or 24 well plate with cold PBS or HBSS (no Ca/Mg), place on ice
2. Euthanize mouse via institutionally approved methods
3. Open peritoneal cavity, expose bile duct and gallbladder (GB)
4. Harvest GB and duct by cutting the duct at duodenal junction and carefully lifting up towards the diaphragm          
5. Place on PBS soaked papertowel and carefully scrape off any pancreas/liver/blood vessels with back of curved scissors or forceps                   
6. Cut open to drain bile, place in prepared plate               
7. Add DTT to prewarmed buffer 1, shake to mix
8. Put 1 ml of buffer 1 into 5 ml Eppendorf tubes
9. Add GB/duct tissue (depending on purposes, can add up to 3 pieces of tissue for same genotype)
10. Incubate at 37 C with shaking at 80 RPM for 10 minutes
11. Using p1000 pipet, gently draw tissue up and down to agitate
12. Decant buffer 1, do not save
13. Add 1 ml prewarmed buffer 2, incubate at 37 C with shaking at 80 RPM for 20 minutes
14. Prepare clean 5 ml Eppendorf tube with 1 ml of FACS wash buffer (FWB see below)
15. Vortex tissue in buffer 2, pipet with p1000 several times. Remove 1 ml supernatant to new 5 ml tube with FWB
16. Add 1 ml prewarmed buffer 2, incubate at 37 C with shaking at 80 RPM for 20 minutes
17. Repeat steps 15 and 16 
    1. Leave this tube on ice or spin down 5 minutes 1300-1500 rpm (4 C), resuspend pellet in 1 ml FWB, and leave on ice
18. Add 1 ml buffer rinse to the tissue and incubate at 37 C with shaking at 80 RPM for 5-10 minutes
19. While incubating in buffer rinse, prepare the digest solution by adding DNase I and Liberase TM
20. Add 1 ml of digest buffer (containing enzymes) to a Miltenyi GentleMACS C tube*
    1. Add tissue, mine with scissors
    2. Add 3 mls additional digest buffer
21. Incubate at 37 C with shaking at 120 RPM for 20-30 minutes, then run “intestine” program on Miltenyi OctoMACS tissue dissociator*
    1. Briefly spin tissue down at 1000 rpm for 1-2 minutes
22. Decant tube through 100 uM filter over clean 15 or 50 ml tube
    1. Wash out C tube with 4-5 mls of FWB
    2. These tubes can be reused several times – save to rinse in water and dry between uses
23. Pipet the cells recovered from EDTA washes in step 17 through the 100 uM filter (combining epithelial cells with cells recovered after digest)
24. Use flat end of 1 ml plastic syringe to mash any visible tissue through the filter, and rinse filter with 4 ml FWB
25. Pellet cells by spinning down 5 minutes 1300-1500 rpm (4 C)
    1. Cell pellet for 1 GB/duct from mouse will be very small
26. Discard supernatant and resuspend in residual FWB (approximately 150 ul) to proceed with staining for flow cytometry

Stock solutions used

DTT = 1M, prepared in water

EDTA = 500 mM, purchased

HEPES = 1 M, purchased

DNase = 100 mg/10 ml H2O

Liberase TM = 50 mg/10 ml H2O

FWB = 500 ml PBS, 2-5 % FBS, 0.1% sodium azide (preservative)

* in the absence of tissue dissociator, mince in 5 ml tube, incubate with shaking and vortex aggressively periodically