In vitro co-culture and 3D matrigel/collagen culture assays

3D Matrigel/Collagen In Vitro Splenocyte Culture Assays

1. Prepare rat tail collagen I mixture by mixing collagen I (Corning), 10x PBS, 0.1N NaOH, H2O, and Matrigel (Corning) in 1.5 ml centrifuge tube to a final collagen concentration of 2 mg/ml with an example mixture below all on ice:

2. Mix collagen solution with Matrigel at a 1:1 mixture (e.g. 500 ul Matrigel using the mixture above), vortex quickly, and place on ice.

3. Coat 200 ul of Matrigel:Collagen mixture or Matrigel alone in each well of a 24-well plate.

4. Seed 250,000 isolated splenocytes in RPMI 1640 + 10% FBS into each coated well or in suspension in uncoated wells with treatment and culture time dependent upon the specific experiment.

5. Following experiment completion, use a pipette to mix and transfer splenocytes with matrices to a 1.5 ml centrifuge tube.

6. Add 1 ml of 2 U/mL dispase II (Roche) and 0.5% w/v collagenase type I (Thermo Fisher) dissolved in RPMI 1640 to each tube containing splenocytes (must be performed even for control splenocytes in suspension culture without matrices) and shake or rock at 37^oC for 10-15 minutes.

7. After digestion, filter splenocytes through 5mL round bottom polystyrene FACS tubes with 35 um strainer cap to remove any excess matrices.  Repeat this procedure with new strainer cap if collagen or Matrigel remain.

8. Transfer splenocytes from polystyrene tubes to new 1.5 ml centrifuge tubes and centrifuge cells at 800 g for 5 minutes.

9. Remove supernatant and wash cells by resuspending splenocytes in 1 ml FACS buffer (PBS + 2% FBS + 1 mM EDTA) and spin down at 800 g for 5 minutes.

10. Remove supernatant and resuspend cells in 200 ul FACS buffer containing antibody dilutions according to manufacture protocol or optimized protocol (Flow cytometry antibody clones and dilutions found in Supplementary Materials) and incubate for 30 minutes to 1 hour at room temperature.

11. Wash cells with 1 ml FACS buffer, centrifuge at 800 g for 5 minutes, remove supernatant, and fix cells with 1% paraformaldehyde for 30 minutes at room temperature and permeabilize cells in permeabilization buffer (BD Biosciences) for 30 minutes if necessary for intracellular stains.

12. Wash cells as before, remove supernatant, and resuspend cells in 500 ul to 1 ml FACS buffer for flow cytometry analysis.  Prior to flow cytometry, labeled splenocytes were filtered into a Falcon 5 mL round-bottom flow tubes with a cell-strainer cap (Corning) to remove residual matrices.

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