7. 24-Well tissue culture treated plates.
8. LumaCyte 96-well plate (LumaCyte WP3110).
9. LumaCyte Radiance instrument.
2.2 In-Process
Infectivity Assay
with Sf9 Cells
(Real-Time Titer)
1. Cell culture medium (see Note 2).
2. Viral stock of known titer (see Note 3).
3. LumaCyte Stabilization Fluid (LumaCyte SF1800).
4. Paraformaldehyde solution (optional—see Note 4): 4% in
0.1 M phosphate buffered saline, pH 7.4.
5. 125 mL shake flasks.
6. 6-Well plates.
7. LumaCyte 96-well plate (LumaCyte WP3110).
8. LumaCyte Radiance instrument.
2.3 Viral
Neutralization Assay
with Vero Cells
1. Dulbecco’s phosphate buffered saline without calcium and
magnesium (DPBS; 1Â).
2. Cell dissociation reagent such as TrypLE or Accutase (see Note
1).
3. Cell culture medium (see Note 2).
4. Viral stock of known titer (see Note 3).
5. LumaCyte Stabilization Fluid (LumaCyte SF1800).
6. Paraformaldehyde solution (optional—see Note 4): 4% in
0.1 M phosphate buffered saline, pH 7.4.
7. 24-Well tissue culture treated plates.
8. LumaCyte 96-well plate (LumaCyte WP3110).
9. LumaCyte Radiance instrument.
3 Methods
The following sections provide example protocols for developing
an infectivity assay with HEK-293 cells, an in-process or real-time
infectivity assay with Sf9 cells, and a viral neutralization assay with
Vero cells. Each of the three assays can be used with different cell
types, viruses, and growth regimes (i.e., adherent vs. suspension),
by adapting these protocols as needed.
3.1 Infectivity Assay
Replacement
with Adherent
HEK-293 Cells (Initial
Titer)
1. Maintain a working stock of cells using standard cell culture
techniques. It is important to monitor the growth and passage
number in order to ensure consistent results.
2. On the day of seeding, detach cells from their growth vessel
using standard techniques for that particular cell line and the
manufacturer’s instructions for the dissociation reagent. For
578
Colin G. Hebert et al.
8. LumaCyte 96-well plate (LumaCyte WP3110).
9. LumaCyte Radiance instrument.
2.2 In-Process
Infectivity Assay
with Sf9 Cells
(Real-Time Titer)
1. Cell culture medium (see Note 2).
2. Viral stock of known titer (see Note 3).
3. LumaCyte Stabilization Fluid (LumaCyte SF1800).
4. Paraformaldehyde solution (optional—see Note 4): 4% in
0.1 M phosphate buffered saline, pH 7.4.
5. 125 mL shake flasks.
6. 6-Well plates.
7. LumaCyte 96-well plate (LumaCyte WP3110).
8. LumaCyte Radiance instrument.
2.3 Viral
Neutralization Assay
with Vero Cells
1. Dulbecco’s phosphate buffered saline without calcium and
magnesium (DPBS; 1Â).
2. Cell dissociation reagent such as TrypLE or Accutase (see Note
1).
3. Cell culture medium (see Note 2).
4. Viral stock of known titer (see Note 3).
5. LumaCyte Stabilization Fluid (LumaCyte SF1800).
6. Paraformaldehyde solution (optional—see Note 4): 4% in
0.1 M phosphate buffered saline, pH 7.4.
7. 24-Well tissue culture treated plates.
8. LumaCyte 96-well plate (LumaCyte WP3110).
9. LumaCyte Radiance instrument.
3 Methods
The following sections provide example protocols for developing
an infectivity assay with HEK-293 cells, an in-process or real-time
infectivity assay with Sf9 cells, and a viral neutralization assay with
Vero cells. Each of the three assays can be used with different cell
types, viruses, and growth regimes (i.e., adherent vs. suspension),
by adapting these protocols as needed.
3.1 Infectivity Assay
Replacement
with Adherent
HEK-293 Cells (Initial
Titer)
1. Maintain a working stock of cells using standard cell culture
techniques. It is important to monitor the growth and passage
number in order to ensure consistent results.
2. On the day of seeding, detach cells from their growth vessel
using standard techniques for that particular cell line and the
manufacturer’s instructions for the dissociation reagent. For
578
Colin G. Hebert et al.