sufficiently large cell sample in order to determine the titer (see
Note 13).
6. Resuspend the cells in an appropriate volume of Stabilization
Fluid in order to achieve the optimal cell count of
~600,000 cells/mL.
7. Add 200 μL of each sample to the LumaCyte 96-well plate.
Place the plate in the Radiance instrument for 15 min before
initiating analysis. Please refer to the Radiance +Autosampler
Quick Start Guide or User Manual for additional details on
operating the Radiance instrument and data analysis. The Radiance measurements can be correlated with the titer of the
supernatant or cell samples taken at the same time after they
have been quantified using comparative methods.
8. The Radiance software can be programmed to provide virus
titer measurements in subsequent assays.
3.3 LFC Viral
Neutralization Assay
with Vero Cells
1. Prior to developing a neutralization assay with LFC, it is necessary to develop an infectivity assay following the procedure
described in Subheading 3.1. This will allow for the optimization of MOI and harvest time. These parameters should be
selected to maximize the dynamic range of the assay in order to
quantify the largest possible range of serum neutralizing
concentrations.
2. 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.
3. 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
example, in the case of Vero cells (ATCC CCL-81) and TrypLE
cultured in a T-75 flask:
(a) Aspirate the cell culture medium and gently wash the cell
monolayer with 5 mL of DPBS.
(b) Remove the DPBS and add 3 mL of TrypLE.
(c) Incubate for 5 min at 37
C or until the cells have visibly
detached.
(d) If needed, gently tap the vessel to dislodge the cells.
(e) Add an appropriate volume of cell culture medium to
resuspend the cells, pipetting up and down to break up
any clumps that are present.
4. Once the cells are resuspended, perform a count in order to
determine the dilution required.
5. Seed cells at 125,000 cells/well in a 24-well plate (see Note
14).
Viral Infectivity Analysis Using Laser Force Cytology
581
Note 13).
6. Resuspend the cells in an appropriate volume of Stabilization
Fluid in order to achieve the optimal cell count of
~600,000 cells/mL.
7. Add 200 μL of each sample to the LumaCyte 96-well plate.
Place the plate in the Radiance instrument for 15 min before
initiating analysis. Please refer to the Radiance +Autosampler
Quick Start Guide or User Manual for additional details on
operating the Radiance instrument and data analysis. The Radiance measurements can be correlated with the titer of the
supernatant or cell samples taken at the same time after they
have been quantified using comparative methods.
8. The Radiance software can be programmed to provide virus
titer measurements in subsequent assays.
3.3 LFC Viral
Neutralization Assay
with Vero Cells
1. Prior to developing a neutralization assay with LFC, it is necessary to develop an infectivity assay following the procedure
described in Subheading 3.1. This will allow for the optimization of MOI and harvest time. These parameters should be
selected to maximize the dynamic range of the assay in order to
quantify the largest possible range of serum neutralizing
concentrations.
2. 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.
3. 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
example, in the case of Vero cells (ATCC CCL-81) and TrypLE
cultured in a T-75 flask:
(a) Aspirate the cell culture medium and gently wash the cell
monolayer with 5 mL of DPBS.
(b) Remove the DPBS and add 3 mL of TrypLE.
(c) Incubate for 5 min at 37
C or until the cells have visibly
detached.
(d) If needed, gently tap the vessel to dislodge the cells.
(e) Add an appropriate volume of cell culture medium to
resuspend the cells, pipetting up and down to break up
any clumps that are present.
4. Once the cells are resuspended, perform a count in order to
determine the dilution required.
5. Seed cells at 125,000 cells/well in a 24-well plate (see Note
14).
Viral Infectivity Analysis Using Laser Force Cytology
581