| Serum Dilutions—using single-channel in 8 well chamber slide |
Serum Dilutions—using multi-channel and 96-well plate |
| Incubate serum with RABV CVS-11 for 90 min |
Incubate serum with RABV ERA NLS-hMGFP for 90 min |
| Add MNA or BSR cells |
Add BSR cells |
| Incubate for 20 h at 37°C |
Incubate for 20 h at 37°C |
| Fixed with acetone for 30 min |
Fixed with 4% formaldehyde for 15 min |
| Stain with anti-rabies FITC antibody containing Evans Blue for 30 min |
Stain with DAPI for 10 min |
| Manual observation of slides with fluorescent microscope |
Automatic quantification using ArrayScan instrument and software |
| Count 20 random fields for presence of RABV infection—about 30–40 min to read five 8-well slides (5 samples, 8 dilutions each) |
Count DAPI and GFP positive nucleus for the entire well—about 30–40 min to read one 96-well plate, 5 samples with 8 dilutions each in duplicates |
| Calculation of titer using Reed–Muench method based on number of fluorescent foci fields at different dilutions |
Calculation of titer using Reed–Muench method based on percentage of GFP positive cells (infected cells) at different dilutions |
