We want to phenotype all leucocyte populations in human tonsils by multi-color flow cytometry.
Do we need an enzymatic treatment for getting out all the leucocytes (DCs, Plasmacells etc.)?
If so what is the protocol, ie. time and type of enzyme, mechanically disruption?
Seems people use a combination of mechanical/enzymatic digestion and a mix of collagenase and Dnase.
Check the protocol below from Miltenyi.
https://www.miltenyibiotec.com/_Resources/Persistent/11c45d2b94d78082f1acc32ea12f7696ec0381bf/Dissociation%20of%20human%20tonsil%20to%20isolate%20%C2%A0mononuclear%20cells.pdf
Here is a paper where they have compared different dissociation methods on tonsil tissue and also done immune phenotyping by Flow Cytometry.
Article Single cell analysis of human tissues and solid tumors with ...
Our lab processes rhesus tonsils and it's a very straightforward protocol for us. We mainly look at natural killer cells and sometimes t cells and this method has worked for us.
Materials:
RPMI 1640 + 5% FBS + 1% penstrep
70 um nylon mesh filters
10 mL luer lock syringe
50 mL falcon tube
Petri dishes
Forceps and scissors
Protocol:
1. Transfer tissue to petri dish and add some media to cover tissue
2. Trim excess fat from tissue and discard
3. Cut tissue into smaller pieces
4. Place tissue on top of 70 um filter that is placed on a 50 mL falcon tube
5. Using the blunt end of the 10 mL syringe, mash tissue through strainer. Wash strainer with media as needed
6. After all tissue is mashed and strainer washed of remaining cells: centrifuge, resuspend, and count.
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