TITLE: Isolation of live single cells from intestinal biopsy
AUTHORS: Tatiana Karakasheva, Kathryn Hamilton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes dissociation of a human intestinal biopsy tissue into single cells, followed by depletion of dead cells via annexin V MACS beads. The outcome is a single-cell suspension with viability ≥ 90% that is used for single-cell sequencing or establishemt of enteroid/colonoid culture.</div></div>

[STEPS]
?. [Before you begin]
Prepare reagents:  Cryo-wash (4.5mL PBS + 500μL FBS) Enzyme Mix (30μL Liberase TH stock + 30μL DNAse I stock + 3mL HBSS) Blocker Buffer (4% BSA in PBS: reconstitute 400mg BSA in 10mL PBS, filter-sterilize) Cell Reconstitution Buffer (2mL Advanced DMEM:F12 + 50μL Blocker Buffer + 20μL DNAse I) 1x MACS Binding Buffer (1mL 20x concentrate + 19mL sterile ddH2O* )
5 mL
1.5 mL
10 mL
2 mL
20 mL
*Do not substitute with deionized water (this will impair annexin V binding)
?. [Recover the cryopreserved tissue]
Thaw the tissue quickly in a  water bath
37 °C
If using a bead bath, put a beaker with pre-heated water into the beads and place the cryovial in the water
?. [Recover the cryopreserved tissue]
As soon as the vial contents are liquified, transfer the tissue suspension to an eppendorf tube
We finely mince the biopsy tissue before cryopreservation. If you are working with a biopsy that was cryopreserved whole, do not skip step 6
?. [Recover the cryopreserved tissue]
Spin the tube 30s - 700g ,  discard the supernatant
Centrifuge: 700 34, 30 s
0 Room temperature
?. [Recover the cryopreserved tissue]
Wash the tissue (repeat 3 times)
?. [Recover the cryopreserved tissue]
Resuspend the pellet in   Cryo-wash
1 mL
?. [Recover the cryopreserved tissue]
Spin the tube 30s -  700g,  discard the supernatant
Centrifuge: 700 34, 30 s
0 Room temperature
?. [Recover the cryopreserved tissue]
Skip this step if biopsy was minced before cryopreservation
?. [Recover the cryopreserved tissue]
Reconstitute the washed tissue in  Cryo-wash supplemented with   DNAse I
500 µl
5 µl
?. [Recover the cryopreserved tissue]
Mince the tissue inside the eppendorf tube using sterile surgical scissors
?. [Recover the cryopreserved tissue]
Spin the tube 30s -  700g,  discard the supernatant
Centrifuge: 700 34, 30 s
0 Room temperature
?. [Single cell dissociation]
Resuspend the pellet in  Enzyme Mix
1 mL
?. [Single cell dissociation]
Incubate in a thermomixer 37oC - 15m, 800RPM
37 33
?. [Single cell dissociation]
During digestion, place a 100μm strainer in a 50mL conical and pre-wet the membrane with with  Blocker Buffer
5 mL
?. [Single cell dissociation]
After digestion, let the tissue fragments settle by gravity to the bottom, pass the supernatant through prepared strainer from step 8.1
?. [Single cell dissociation]
Repeat the digestion (steps 7-9) with fresh Enzyme Mix 2 more times (3 cycles of 15m total)
?. [Single cell dissociation]
After the final digest, pass the tissue fragments 10 times through a P1000 tip
?. [Single cell dissociation]
Pass the digested suspension through the strainer:
?. [Single cell dissociation]
Apply the suspension from step 11 directly to the strainer membrane
?. [Single cell dissociation]
Take the strainer out of the conical tube and place it in a round 30mm TC dish
A larger size dish can be used in lieu of a 30mm dish
?. [Single cell dissociation]
Use the flat surface of an insulin syringe plunger to force the remaining undigested fragments through the strainer by rubbing the plunger in a circular motion, discard the plunger
?. [Single cell dissociation]
Rinse the strainer by lifting it slightly from the round dish and applying  Blocker Buffer, capturing the flow-through in the 30mm dish; keep the strainer suspended
2 mL
?. [Single cell dissociation]
Rinse the strainer bottom with  Blocker Buffer, capturing it in the 30mm dish
2 mL
?. [Single cell dissociation]
Transfer the suspension from the 30mm dish to the 50mL conical from step 8.1
?. [Single cell dissociation]
Rinse the 30mm dish with  Blocker Buffer and transfer to the same conical
1 mL
?. [Single cell dissociation]
Transfer the suspension from 50mL conical to a new 15mL conical.
This step is optional, but recommended, because it results in a more visible pellet than when spinning in a 50mL conical
?. [Single cell dissociation]
Spin down the cells 5m - 500g, resuspend in  Cell Reconstitution Buffer
1 mL
Centrifuge: 500 34, 5 min
0 Room temperature
?. [Single cell dissociation]
Strain the suspension into a FACS tube through the 35μm strainer cap
It is essential to strain the cells before proceeding to column-based dead cell removal
?. [Single cell dissociation]
Count the cells, noting the viability
?. [Dead cell removal]
Pellet the cells: 5m - 500g, discard the supernatant
Centrifuge: 500 34, 5 min
0 Room temperature
?. [Dead cell removal]
Resuspend the pellet in  of MACS Dead Cell Removal MicroBeads
200 µl
?. [Dead cell removal]
Incubate 15m - room temperature
0 Room temperature
?. [Dead cell removal]
During the incubation, prepare the MACS LS column by placing it into the magnet and rinsing with  MACS Binding Buffer (discard the flow-through)
3 mL
?. [Dead cell removal]
Add  MACS Binding Buffer to the suspension from step 19 to total volume of
300 µl
500 µl
?. [Dead cell removal]
Apply the suspension to the prepared MACS LS column (in the magnet) and collect the flow-through into a 15mL conical tube
The flow-through contains live cells, while dead cells are captured in the column
?. [Dead cell removal]
Wash the column 4 times with  MACS Binding Buffer, keep collecting the flow-through (live cells) into the same tube.
3 mL
At this point the column can be discarded, unless you are troubleshooting and intend to elute the captured dead cells.
?. [Dead cell removal]
Spin the live cell suspension 5m - 500g, discard the supernatant
Centrifuge: 500 34, 5 min
0 Room temperature
?. [Dead cell removal]
Resuspend the pellet in  Cell Reconstitution Buffer
500 µl
?. [Dead cell removal]
Count the cells, noting the viability