Mike Barnkob:Protocols/Immunology/Enrichment from tumours

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T cell enrichment from tumours

Protocol on how to isolate T cells from in vivo tumours using Percoll seperation.

I find that the Percoll seperation is good for larger tumours with lots of dead cells (for example later stages of B16.F10 tumours). For smaller tumours, Percoll seperation it not as necessary.

Updated 27-7-15.

Reagents and equipment

For harvesting tumour and lymph nodes:

1. Petri-dish.
2. R10 media.
3. 70% ethanol.

For single-cell suspension:

1. 50 ml Falcon tube
2. 70μM cell strainer
3. 2.5ml syringe
4. R10 T cell media.

For Percoll seperation:

1. "100%" Percoll: Add 100 ml 10x PBS to 900 ml Percoll.
2. Prepare dilutions of 80%, 50% and 40% solutions of "100%" Percoll, with 4 ml per dilution per sample.

For T-cell enrichment:

1. Antibodies for FACS.

Protocol

Harvest tumour:

1. Euthanize mouse.
2. Dissect out tumour and spleen
   Note: Wet large area of skin with 70% ethanol to disinfect.
   Note: Keep hair out of incision.
3. Using forceps and scissor, dissect out tumour.
   Note: remove as much surrounding fat and skin as possible, but keep skin on top.
4. Place tumour in petri dish and weigh.
5. Cut tumours into smaller pieces (2-4mm) and transfer into tube with 5ml R10 media. Keep on ice.
6. Dissect out spleen and transfer into tube with 5ml R10 media. Keep on ice.

Create single-cell suspension:

1. Add 1mL of R10 T cell media to 50 ml Falcon tube. Place 70μM cell strainer on tube and transfer tumour onto cell strainer.
   Note: Work in tissue-culture hood.
2. Take out plunger of 2.5ml syringe, and mesh tumour through strainer with blunt end of the plunger.
   Note: Flush cells through with a little media afterwards.
   Note: When done, add up to 10-15 ml R10 T cell media total.
3. Spin cells down at 1500 RPM for 5 min and remove supernatant.
4. Optional for tumours: Resuspend in 2 ml of red blood cell lysis buffer and leave on ice for 3 min. Add 5 ml of media, spin cells down at 1500 RPM for 5 min and remove supernatant.

Percoll seperation:

1. Create Percoll gradient: Add 80% solution to bottom of 15ml Falcon tube. Slowly add 50% solution on top.
   Note: I normally use 4 ml per gradient.
2. Resuspend samples in the 40% solution, and very slowly add to 80%-50% tubes. Draw lines on the tubes of where the gradients are.
3. Spin samples at 400g for 30 minutes with lowest speed-up and speed-down speeds.
4. Using 5ml pipet, transfer dead cells from top of samples and discard.
5. Using Pastur-pipet aspirate interfase at 80%-50% border and transfer to new 15ml Falcon tube.
   Note: Starting with the spleen sample, it should be easy to see where the lymphocyte population is. If nothing is visible, aspirate the whole 50% gradient.
6. Wash cells in either FACS buffer or R10 for T-cells media and count.

Futher processing:

1. Stain cells for FACS or bead-seperation.

References

• Tissue Dissociation Guide: http://www.worthington-biochem.com/tissuedissociation/Tumor.html
• gentleMACS tumour dissociation protocol: http://www.miltenyibiotec.com/~/media/Files/Navigation/Sample%20preparation/Miltenyi%20Protocols/Tumor_Dissociation_mouse.ashx