TITLE: Freezing and processing intestinal biopsies for the isolation of CD45+ leukocytes
AUTHORS: Oxford HCA, Nicholas Provine, Michael FitzPatrick, Melanie Dunstan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is designed for the freezing, thawing, and processing of human intestinal pinch biopsies for isolation of live CD45+ leukocytes and downstream applications such as flow cytometry or single-cell RNA-sequencing.</div></div>

[STEPS]
?. [Stage 1 - Collection in Endoscopy]
Collect endoscopic biopsies into 50ml falcon tube with 5ml of cold MACS tissue storage solution.
?. [Stage 1 - Collection in Endoscopy]
Ensure all biopsies are immersed in the solution.
?. [Stage 1 - Collection in Endoscopy]
Keep on ice until ready to freeze.
?. [Stage 1 - Collection in Endoscopy]
Move onto Stage 2 as soon as possible, within 6 hours maximum.
?. [Stage 2 - Freezing samples]
Perform all stages in the hood.
?. [Stage 2 - Freezing samples]
Ensure that a CoolCell LX cell freezing container is available, and is defrosted to room temperature.
?. [Stage 2 - Freezing samples]
Label a cryovial for each sample, as appropriate.
?. [Stage 2 - Freezing samples]
Add 1000μl of Cs10 to cryovial.
?. [Stage 2 - Freezing samples]
Carefully remove all MACS tissue storage solution from the 50ml falcon with a pastette (transfer pipette) and P1000 pipette, taking care not to disrupt or discard the biopsies.
?. [Stage 2 - Freezing samples]
Using sterile forceps, transfer the biopsies into the Cs10-containing cryovial.
?. [Stage 2 - Freezing samples]
Place cryovials into CoolCell LX and transfer to -80 C freezer.
?. [Stage 2 - Freezing samples]
Move to Stage 3 on the next working day if possible.
?. [Stage 3 - transfer samples to storage location]
Remove sample from CoolCell LX and transfer to liquid nitrogen box (if for long-term storage), or to -80 C box (if planned for use in next 2 months).
?. [Stage 4 - thawing and digesting biopsies]
Perform all stages in the hood.
?. [Stage 4 - thawing and digesting biopsies]
Warm Complete Media and Digestion Media to 37 C prior to beginning.
?. [Stage 4 - thawing and digesting biopsies]
Proceed quickly to remove the biopsies from the freezing media.
?. [Stage 4 - thawing and digesting biopsies]
Fill a gentleMACS C tube with 5 ml of Digestion Media.
?. [Stage 4 - thawing and digesting biopsies]
Fill an appropriately labelled 15 ml conical with 9 ml of Complete Media.
?. [Stage 4 - thawing and digesting biopsies]
Begin to thaw the frozen biopsy in a 37 C waterbath.
?. [Stage 4 - thawing and digesting biopsies]
When the frozen biopsy begins to thaw, but haven’t thawed completely, remove from the water bath and transfer to the hood.
?. [Stage 4 - thawing and digesting biopsies]
Finish thawing the biopsy by adding 1 ml of Complete Media (from the 15 ml conical) dropwise.
?. [Stage 4 - thawing and digesting biopsies]
Use a pastette to transfer the media and biopsies into the appropriate 15 ml conical.
?. [Stage 4 - thawing and digesting biopsies]
Place a 70 micron cell strainer onto a 50 ml conical.
?. [Stage 4 - thawing and digesting biopsies]
Pour the biopsies onto the strainer.
?. [Stage 4 - thawing and digesting biopsies]
Wash with 20 ml of Complete Media.
?. [Stage 4 - thawing and digesting biopsies]
Use sterile forceps to transfer the biopsies into a gentleMACS C tube filled with 5 ml of Digestion Media.
?. [Stage 4 - thawing and digesting biopsies]
Homogenize on the gentleMACS using programme brain 01_02 (gentle).    Tissue tends to get caught in the rotor blade of the gentleMACS tube, use pastette to return to bottom of tube.
?. [Stage 4 - thawing and digesting biopsies]
Place in a 37 C shaking incubator (220 rpm) for 1 h.
?. [Stage 4 - thawing and digesting biopsies]
Homogenize on the gentleMACS using programme m_intestine_01.
?. [Stage 4 - thawing and digesting biopsies]
Strain cells through 70µm filter into 50mL falcon tube. Pour onto filter.
?. [Stage 4 - thawing and digesting biopsies]
Grind remaining tissue (use plunger from a syringe to break up any clumps on top of filter (pestle and mortar-action).    Wash any cells around blades, on filter or still in tube using Complete Media and a pastette.
?. [Stage 4 - thawing and digesting biopsies]
Centrifuge at 600 g for 10 minutes at 4 C.
?. [Stage 4 - thawing and digesting biopsies]
Resuspend in 20 ml of Complete Media.
?. [Stage 5 - Lymphocyte enrichment]
Centrifuge at 600 g for 5 minutes at 4 C.
?. [Stage 5 - Lymphocyte enrichment]
Resuspend cell pellet in 6mL of 35% Percoll.
?. [Stage 5 - Lymphocyte enrichment]
Transfer to 15mL falcon tube.
?. [Stage 5 - Lymphocyte enrichment]
Underlay 3 mL of 70% Percoll solution using a glass Pasteur pipette.
?. [Stage 5 - Lymphocyte enrichment]
Centrifuge at 800xg for 20 minutes (at RT) without the brake (decrease acceleration to 7).
?. [Stage 5 - Lymphocyte enrichment]
Take the layer between 35% and 70% Percoll and transfer to a new 15mL falcon tube.    Before removing interphase, remove some of the top layer to avoid stromal cell contamination of mononuclear fraction.
?. [Stage 5 - Lymphocyte enrichment]
Fill the tube to 15 ml with Complete Media.
?. [Stage 5 - Lymphocyte enrichment]
Centrifuge at 600xg for 5 minutes.
?. [Stage 5 - Lymphocyte enrichment]
Remove supernatant.
?. [Stage 5 - Lymphocyte enrichment]
Resuspend cell pellet in 10mL Complete Media.
?. [Stage 5 - Lymphocyte enrichment]
Count cells using trypan blue.
?. [Stage 5 - Lymphocyte enrichment]
Cells are now ready for downstream applications