Ex vivo tissue cytokine analysis

Ex vivo tissue cytokine analysis

Materials:

Medium: RPMI Medium 1640 + 10% FBS + PSQ

FACS buffer: 3 % FBS and 2 mM EDTA in PBS

Procedure:

1. Collect Spleens and tumors and store in medium on ice
2. All following steps were performed on ice or at 4 ° C unless noted differently
3. Process tumor into single cell suspension using Miltenyi mouse tumor dissociation kit following the manufacturer’s protocol 
4. Process spleen into single-cell suspension using gentle MACS mechanical dissociation (spleen 4 program)
5. Mash tissue through a 70-micron filter using the rubber end of a 3 ml syringe. 
6. Pellet cells by centrifugation at 1200 rpm for 5 minutes. 
7. Lyse Red blood cells by resuspending the cell pellet in 1 ml ACK lysis buffer and incubate on ice for 5 minutes. Quench ACK lysis buffer with FACS buffer. 
8. Pellet cells by centrifugation at 1200 rpm for 5 minutes. 
9. Resuspend cell pellet in 1 ml of medium
10. Plate 0.2 ml in 96-well plates (20,000 - 400,000 tumor cells or 50,000 -70,000 spleen cells). 
11. Incubate cells overnight at 37 ° C
12. Collect culture supernatant 
13. Perform cytokine analysis using V-Plex Proinflammatory MSD kit following manufacturer protocol (Meso Scale Diagnostics K15048D-4). 
14. Normalize the concentrations of cytokines to the number of cells plated.