Immune Netw.  2017 Oct;17(5):307-316. 10.4110/in.2017.17.5.307.

Semi-Functional Quantitative Flow Cytometry Assay for Lymphocytic Choriomeningitis Virus Titration

• Ban YH
• Ha SJ

• KMID: 2400636
• DOI: http://doi.org/10.4110/in.2017.17.5.307

Abstract

Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.

Keyword

Lymphocytic choriomeningitis virus; Semi-functional quantitative flow cytometry assay; Virus plaque assay; Nucleoprotein

MeSH Terms

Animals
Antibodies
Flow Cytometry*
Humans
Lymphocytic choriomeningitis virus*
Lymphocytic Choriomeningitis*
Methods
Mice
Nucleoproteins
Polymerase Chain Reaction
Antibodies
Nucleoproteins

Figure

• 

  Figure 1 Vero cells were more susceptible than BHK21 cells to LCMV ARM infection. A total of 3.0×105 Vero and BHK21 cells were plated in a 6-well plate with 3 ml of DMEM complete medium for 15 h before use. After the removal of the culture medium, cells were treated with 200 µl of serial dilutions (106 to 102 pfu/ml) of LCMV ARM for 1 h. A total of 2 ml of the medium was dispensed and cells were stained with VL-4 after 24 h of incubation, followed by flow cytometry analysis. (A) NP expression in Vero and BHK21 cells infected with serial dilutions of LCMV ARM. (B) The expression of isotype control of VL-4 antibody (IgG2a) in Vero and BHK21 cells infected with LCMV ARM (105 pfu/ml). The number in the plot indicates the percentage of NP+ cell population in Vero and BHK21 cells. Data are representative of 5 independent experiments.SSC-A, side scatter area.

• 

  Figure 2 Initial cell seeding number was set to high and low for each plate scale. Cell numbers for each scale were calculated in proportion to the size of 6-, 12-, and 24-well plate. Cells were seeded and incubated for 15 h prior to infection. The number in the image visualized by an optical microscope indicates the cell count of seeded cells as high (top) and low (bottom) confluence in each plate. Data are representative of 2 independent experiments.

• 

  Figure 3 Validation of conditions such as plate scale, cell seeding number, and incubation time to establish an optimal method for SFQF assay. Vero cells were plated on 6-, 12-, and 24-well plates at high and low seeding numbers (as previously determined) and cultured for 15 h. The medium was removed and cells were treated with 200 µl of the serial dilution of LCMV ARM stock (106 pfu/ml) at each incubation time, followed by flow cytometry analysis. (A) The incubation of 48 h in a 6-well plate seeded with a high number of cells was optimal for LCMV detection. Numbers in representative plots indicate percentages of NP+ cells at indicated conditions such as cell seeding number and incubation time (left top, high and 24 h; left bottom, low and 24 h; right top, high and 48 h; right bottom, low and 48 h). (B) Line graphs summarize the representative data of Fig. 3A obtained from each condition. Data are representatives of 2 independent experiments.SSC-A, side scatter area.

• 

  Figure 4 Plaque numbers and NP+ cell frequencies were correlated to obtain a standard curve. Vero cells were plated as per the conditions in Fig. 3 and infected with 200 µl of 2-fold dilution of LCMV ARM (106 pfu/ml) in the amount same as that in the plaque assay. After 48 h of incubation, the frequency of NP+ cells was analyzed by flow cytometry. All of the serial dilutions were analyzed by plaque assay to verify the threshold of the virus detection. Calibration curve of NP+ cell frequency (yellow circle) and plaque number (green circle). Calculation of plaque assay for each serial dilution (yellow green circle). Left Y-axis, right Y-axis, and X-axis indicate plaque number with NP+ cell frequency, calculated LCMV titer of each serial dilution, and titer of each treated serial dilution, respectively. R2 values were obtained using non-linear curve fit analysis. Data are representative of 3 independent experiments.

• 

  Figure 5 Validation of LCMV titration in tissues ex vivo. C57BL/6 mice were intravenously infected with LCMV CL13 (2×106 pfu/ml). Spleens and serum were harvested from mice 10 days after infection. Homogenized spleens and bled serum from 10−4 to 10−2 dilution of original stocks were incubated with Vero cells and SFQF and plaque assays were manually performed. (A) Detection of NP+ proportion in serum and spleens of mice infected with LCMV CL13. Numbers in plots indicate the percentage of NP+ population. (B) Bar graphs summarize the titer calculated from the plaque assay (green bar) and frequency of NP+ cells measured by SFQF assay (yellow bar). n=3 mice per group in each experiment.

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