Isolation of renal-infiltrating T cells

Processing Kidneys for FACS

Reagents:

Keep Collagenase D stock at 4000lU/mL (Roche #1108882001)

Collagenase D Digestion mix: 100 lU/mL collagenase D, 10% FBS in RPMI with CaCl2(2mM) and MgCl2(2mM)

(Collagenase D was sold in mg. The manufacturer will test the activity of each batch and listed on the bottle. Please aliquot based on the provided activity in U provided by the manufacturer)

1. Inject IV antibodies (CD45.1/2 PE or Thy1.1/2 PE) 5 minutes before sacrificing the mice to distinguish intra-vascular and extra-vascular cells.
2. Put both kidney into 15mL tube with PBS after sacrificing the mouse
3. Transfer kidney samples into 60mm dish and remove both capsule layers (fibrous tissue) with forceps
4. Place kidney and Coll. D mix (6mL for both kidney) into 60mm dish and smash kidney into small pieces ~15-20 pieces by the bottom of syringe
5. Transfer Coll. D mix with tissue into 15mL tube
6. Incubate for 45 min at 37 °C, shake with 250rpm
7. Transfer the Coll. D mix with cells into gentleMACS tube, run the m_spleen_01_01 program with gentleMACS dissociator
8. Transfer Coll D mix into a new 50mL tube through 70μm filter.
9. Spin down 1600 RPM for 5 min
10. Resuspend pellet with 4mL PBS 
11. Slowly transfer 4mL PBS with cells to 4mL of Ficoll. Suggest to place 15mL tube nearly horizontal, and drop PBS with cell solution along the wall of tube
12. Spin for 20 min at 700g, no brake
13. Carefully remove from centrifuge and collect mononuclear cells at the interphase
14. Take interphase and wash with PBS and spin
15. Resuspend in PBS and stain for FACS 

Even after Ficoll isolation, there are still a lot of cell debris and unwanted cells such as tubular cells. To ensure a more purified population on your gate, I will suggest to double stain the gates, such as TCRb+ CD4+ for the CD4 T cells.