Preparation of retinal cells
1| Bleed mice by aspirating blood from the heart: Insert a 24G needle, on a 1ml syringe, below the rib cage in the center, directed slightly to the left and downwards at an angle of about 15-20o to the surface
2| Enucleate eyes and put in complete medium in Petri dish for immediate retinal isolation.
3| Under a dissecting microscope cut along the limbus of the eye and carefully remove the lens and cornea; peel the retina off carefully and cut off the attachment at the optic nerve.
4| Mince retina tissue in collagenase/DNase digestion buffer
Buffer: Complete medium containing collagenase (1mg/ml) + DNase (10ug/ml)
5| Incubate retina in DNAse buffer at 37°C for 3hrs.
6| Pipette cells intermittently (every 30 or 60 minutes) using 10 ml pipette.
7| After 3 hrs quench digestion reaction with 5-10 fold volume 10% FBS in medium.
8| Fill tube with medium and centrifuge at 1200rpm for 10minutes
9| Wash pellet in complete medium (2X)
10| Count the retinal cells
Retina organ-TH17 Co-culture system:
1| Isolate naive syngeneic CD4+ T-helper cells (>95%)
2| Culture the naïve T cells in medium containing anti-CD3/anti-CD28 Abs under TH17 polarizing condition [TGF-β1 (10ng/ml), IL-6 (10ng/ml) anti-IFNγ (10 µg/ml) and anti-IL-4 (10µg/ml) Abs) for 48 hours
3| Add differentiating TH17 cells to freshly isolated retina cells (1 TH17 cell: 5 retina cells)
4| Propagate co-culture for additional 48 hours under the TH17 polarizing condition in the presence or absence of 10µg/ml anti-IL-27p28 Abs
5| Controls: retinal cells alone; Differentiating TH17 cells alone.
6| Add PMA and Ionomycin for last 5 hours and Golgistop for last 1 hour as described above into the cultures.
7| Perform four-color cell surface/intracellular staining using antibodies against CD4, IL-17, IFN-γ, and CD44 and isotype control antibodies.