TITLE: Dissociation of fresh colorectal biopsies
AUTHORS: Alan Simmons, Ken Lau

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Current procedures for the dissociation of intestinal biopsies (<50mg)</div></div>

[STEPS]
?. [Reciept / Grossing]
Upon reciept, inspect tissue and record information such as:    Accession # and other relevant collection metadata    Size - this can be as simple as drawing a spot the size of the tissue with description (flat, round, etc.)    Appearance    Presence of blood    Weight - in the interest of moving forward and not drying out a sample, we do not weigh tissue. Our samples are typically 20 - 40mg or less
?. [Chelation]
Move tissue to a 2ml tube containing ~1.8ml of chelation buffer and place on a tube rotator with continuous rotation.
?. [Dissociation]
Gently remove chelation buffer and re-suspend tissue in DPBS.
?. [Final washes]
Pipette cells to mix and re-suspend before transfering onto a 70µm filter on a clean tube in a cold rack. Allow cells to filter through by gravity and check filtrate for cells before proceeding.
?. [Reciept / Grossing]
Wash tissue gently in DPBS to remove any debris or excess transport media.
?. [Reciept / Grossing]
If tissue is solid/pedunculated, it may need to be bisected or trisected to increase surface area, do not cut smaller than 2mm2.
?. [Chelation]
Gently replace chelation buffer on sample at 30 minutes and 1 hour into chelation, ensuring that enough air remains in tube to displace (agitate) tissue during rotation
?. [Chelation]
Prepare DNAse for use before chelation is over by adding 1ml of DPBS to a frozen 1ml aliqout of 2x DNAse and leaving on bench to thaw. Move DNAse to Ice after it has thawed.
?. [Dissociation]
Gently remove DPBS from tissue and resuspend in 1.8ml of Dissociation solution. Make sure there is enough air in the tube to displace the tissue on inversion.
?. [Dissociation]
Quickly prepare Dissociation solution by adding 1.9ml of 1x DNAse to a frozen 100µl aliquot of 20x cold Protease. Mix by pipetting.
?. [Prep work]
Hydrate, aliquot, and freeze 2x DNAseHydrate to 5mg/ml in DPBS; keep cold; aliquot 1ml each into 2ml tubes and freeze immediately.Store at -20˚c and use within 6 months.
?. [Prep work]
Hydrate, aliquot, and freeze 20x Cold proteaseHydrate to 100mg/ml in molecular grade H2O; keep cold; aliquot 100ul each into 2ml tubes and freeze immediately.Store at -20˚c and use within 6 months.
?. [Dissociation]
Place on a tube rotator with continuous rotation.
?. [Dissociation]
At 25 minutes (end of dissociation) triturate using a normal p-1000 tip 5-10 times, such that tissue is deformed as it enters the tip to yield material into the supernatant, and clusters are broken apart. The supernatant should become somewhat cloudy with cells. Inspect supernatant on a hemocytometer. Cells shoud be mostly single or in clusters of 10 or less. There should be at least 100K cells total. If tissue does not adequately yield cells, continue incubation for another 5 minutes before triturating again.
?. [Final washes]
Spin cells at 700xg for 5 minutes to pellet.
Centrifuge: 700 1324, 5 min, 4-6 10
?. [Final washes]
Ensure that cells have visibly pelleted before removing supernatant. Resuspend in DPBS using a wide-bore p-1000 tip. If cells do not pellet, you may need to increase speed by 100xg. As cells continue to die and secrete material, you may observe stringy aggregates in the supernatant or pellet. Ideally this material remains in the supernatant and is removed during washes, however if persists and becomes too large it may prevent cells from pelleting.
?. [Prep work]
On the day of tissue reciept, prepare chelation buffer:  AB1DPBS49.35ml20.5M EDTA400µl30.1M DTT (diluted from 1M stock)250µl
AB1DPBS49.35ml20.5M EDTA400µl30.1M DTT (diluted from 1M stock)250µl
?. [Prep work]
Dilute to 0.1M, aliquot, and freeze stock DTTDilute the 1M stock to 0.1M by adding 1 part 1M DTT to 9 parts Molecular bio grade H2O. Mix well and aliquot at 1-2ml each. Freeze and store at -20˚c for up to 6months. Use 0.1M aliquots within 1 week of thawing.
?. [Final washes]
Repeat the previous 2 steps (wash) twice, checking cell number and quality upon resuspension.
?. [Final washes]
Check cells, dilute as needed and proceed to encapsulation.
?. [Final washes]
On the 3rd resuspension into DPBS (or single cell running buffer), gently resuspend cells at roughly 2x the desired final concentration or greater and pass through a 70µm flowmi filter into a clean tube.
?. [Final washes]