High-throughput neutralization assay of AAV-GFP vectors using sera

The Celigo Image Cytometer can measure the percentage of GFP+ cells and the mean fluorescent intensity distributions across different dilutions. The ability to rapidly assess serial dilutions gives added confidence to assays that are inherently variable [33]. 

In this experiment, Celigo was used to image and quantify GFP expression after the target cells were treated with different concentrations of sera.

1. The target cells were seeded in a 96-well plate and allowed to adhere overnight
2. Cells were transduced with AAV-A vector at mid-MOI
3. Cells were pre-incubated overnight with different dilutions of serum (1X, 10X, and 100X)
4. The Celigo was used to image and analyze the cells in brightfield and GFP fluorescence to determine transduction efficiencies

Serum samples exerted dose-dependent neutralization effects on transduction efficiencies

The Celigo was able to directly measure the GFP+ percentages in the presence or absence of different concentrations of sera. The GFP fluorescent images and analysis results are shown in Figure 1 and Figure 2 respectively. 

• The GFP fluorescent images clearly showed that 1X diluted serum samples dramatically reduced GFP+ percentages (<1%)
• The graph confirms that the percentage of GFP-expressing cells was slightly higher for the 10X dilution (~5% of cells)
• GFP+ percentages in wells treated with the 100X serum dilution were similar to that of the positive control (16-18% of cells)

Figure 1. GFP fluorescent images for negative and positive controls, sera at 1X, 10X, and 100X dilutions.

Figure 2. GFP+ percentages of target cells demonstrating dose-dependent transduction efficiencies.

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