High-throughput counting of GFP-expressing influenza viral foci for focus formation assay in 96-well plates

The Celigo Image Cytometer can be used to count and analyze fluorescent or colorimetric foci.  The foci are generated when the target virus do not induce lysing of the host cells.  These foci can be detected by using fluorescent viral particles, fluorescent immunostaining, and immunostaining with horse radish peroxidase (HRP). These foci images are typically captured manually using a fluorescent microscope and analyzed using an external image analysis software such as ImageJ.

In this work, the GFP-expressing influenza virus was grown under an overlay of methylcellulose and media, thus enabling the formation of tight fluorescent foci.  The viral solutions were diluted from 300 ffu/well to 4.7 ffu/well in 2-fold dilutions, where a negative control was also prepared without any virus.  The Vero cells were infected with the GFP-expressing influenza virus for 31 hours and fixed before image cytometric analysis.

Celigo Image Cytometer automatically acquired and analyzed whole well fluorescent images for 96-well plates, generating the number of foci per well.  Bright field and fluorescent images can be captured and analyzed in less than 10 min/plate, equivalent to 50 plates/day when integrated with automation.

GFP-expressing influenza viral foci

Whole well bright field and fluorescent overlay image, as well as zoomed-in fluorescent image showing the cluster for fluorescent foci

Below are examples of influenza viral titers with different quantities of fluorescent foci generated in each well.  These fluorescent foci are directly counted in the images, which can be used to generate a viral titer plot to better characterize viral infection.

viral titer at different dilution
viral titer at different dilution

Whole well and zoomed-in fluorescent images of the viral titer at different dilution of the virus for the viral titer assay

foci counting titration

Foci counting results in respect to the FFU infecting the Vero cells

The Celigo Image Cytometer automates viral titer assays: