Tubes were incubated in a heating block at 70 °C for 120 min, with manual shaking every 10 min.
Cells were then cultured at 37°C for 3 h with gentle shaking every 30 min and then washed twice with phosphate-buffered saline (PBS).
Afterwards, DENV-2 supernatant was added, followed by incubation for 1 hr with gentle shaking every 10 min for optimal virus to cells contact.
The warmed HybA media was aspirated, and the retina was incubated with Papain (0.06 mg/ml Worthington biochemical, Lakewood, NJ, USA) in HybA for 20 min at 37 °C in a water bath with gentle shaking every 10 min.
After incubation for 45 min, 160 μL 20% SDS was added and mixed by inversion several times with further incubation at 60°C for 1 h with intermittent shaking every 15 min.
The remaining soil pellets were treated three times with 400 μL extraction buffer and 60 μL SDS (20%) and kept at 60°C for 15 min with intermittent shaking every 5 min.
For immunofluorescence of CTL/target conjugates, EL4 target cells were pulsed with 1 μM OVA peptide in normal EL4 culture medium for 1 h at 37°C, with gentle shaking every 20 min. The pulsed EL4 was washed twice with normal medium.
200 μl of this buffer was added to each pellet, mixed and incubated at 37°C for 90 min with gentle shaking every 30 min. 200 μl of water was added to each well and the plate was centrifuged at 6000 x g for 4 min (for centrifuges with maximum 3,000 x g, centrifugation times can be tripled) and the supernatant was decanted.
Virus was adsorbed to cells for 1 h, with shaking every 15 min. Wells were overlaid with 1.6% w/v Bacto agar (DIFCO, BD Diagnostic Systems, Palo Alto, CA, USA) mixed 1∶1 with L-15 media (Cambrex, East Rutherford, NJ, USA) containing antibiotics and fungizone, with or without 0.6 µg/ml trypsin (Sigma, St . Louis MO, USA).
