using the Cellometer Auto 2000 cell viability counter. Transfer
1 Â 10
6 cells into two tubes, and label one tube “negative
control” and the second “positive control.” Wash cells by
adding 1 mL of PBS to each tube and centrifuging at
500 Â g for 5 min at 4
C. Remove supernatant and fix cells
by adding 100 μL Cytofix™ Fixation Buffer. Fixing the cells
allows the viability dye to pass through the cell membrane and
react with free amines both on the cell surface and in the cell
interior to generate strong fluorescence that is characteristic of
nonviable cells. Mix cells thoroughly and incubate for
15–30 min at 4
C in the dark. Perform the same wash steps
as described previously. Resuspend the negative control cells in
500 μL staining buffer. In contrast, stain the positive control
cells with 50 μL of the working concentration (1:100) of
LIVE/DEAD™ Fixable Dead Cell Stain for 20 min at 4
C
in the dark. Wash and resuspend positive control cells in 500 μL
staining buffer. Lastly, add 150–200 μL of the negative control
cell suspension into the positive control tube. This will allow
simultaneous visualization of the live and dead cell populations
in the positive control tube upon acquisition on the flow
cytometer.
13. The 96-well DeepWell™ polypropylene microplate is ideal for
staining a large set of samples and optimal washing of cells with
buffer volumes of up to 2 mL/well.
14. Remove the supernatant in a round-bottom polystyrene tube
after centrifugation by using a Pasteur pipette attached to a
vacuum aspirator. Alternatively, carefully pour the supernatant
into a waste receptacle. If using a 96-well DeepWell™ polypropylene microplate, quickly invert the microplate over a waste
receptacle and gently tap once on a paper towel to remove
residual volume.
15. Antibody titration must be performed to determine the optimal concentration of each antibody used. This can be determined by looking at the staining index over a range of
concentrations [10]. The aim is to select a concentration that
gives bright staining with minimum background. A starting
concentration of 0.5 μg antibody per 1 Â 10
6 cells in 50 μL
staining buffer can be used for staining optimization.
16. Run the appropriate controls, such as single-stain compensation beads and Fluorescence Minus One (FMO) controls prior
to sample acquisition. Single-stain compensation beads should
be acquired every experiment for optimal correction of fluorochrome spectral overlap. In contrast, FMO controls are cells
that are stained with all the fluorochromes except one fluorochrome. These cells are critical in setting up the cell gating and
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Gerard Agbayani et al.
1 Â 10
6 cells into two tubes, and label one tube “negative
control” and the second “positive control.” Wash cells by
adding 1 mL of PBS to each tube and centrifuging at
500 Â g for 5 min at 4
C. Remove supernatant and fix cells
by adding 100 μL Cytofix™ Fixation Buffer. Fixing the cells
allows the viability dye to pass through the cell membrane and
react with free amines both on the cell surface and in the cell
interior to generate strong fluorescence that is characteristic of
nonviable cells. Mix cells thoroughly and incubate for
15–30 min at 4
C in the dark. Perform the same wash steps
as described previously. Resuspend the negative control cells in
500 μL staining buffer. In contrast, stain the positive control
cells with 50 μL of the working concentration (1:100) of
LIVE/DEAD™ Fixable Dead Cell Stain for 20 min at 4
C
in the dark. Wash and resuspend positive control cells in 500 μL
staining buffer. Lastly, add 150–200 μL of the negative control
cell suspension into the positive control tube. This will allow
simultaneous visualization of the live and dead cell populations
in the positive control tube upon acquisition on the flow
cytometer.
13. The 96-well DeepWell™ polypropylene microplate is ideal for
staining a large set of samples and optimal washing of cells with
buffer volumes of up to 2 mL/well.
14. Remove the supernatant in a round-bottom polystyrene tube
after centrifugation by using a Pasteur pipette attached to a
vacuum aspirator. Alternatively, carefully pour the supernatant
into a waste receptacle. If using a 96-well DeepWell™ polypropylene microplate, quickly invert the microplate over a waste
receptacle and gently tap once on a paper towel to remove
residual volume.
15. Antibody titration must be performed to determine the optimal concentration of each antibody used. This can be determined by looking at the staining index over a range of
concentrations [10]. The aim is to select a concentration that
gives bright staining with minimum background. A starting
concentration of 0.5 μg antibody per 1 Â 10
6 cells in 50 μL
staining buffer can be used for staining optimization.
16. Run the appropriate controls, such as single-stain compensation beads and Fluorescence Minus One (FMO) controls prior
to sample acquisition. Single-stain compensation beads should
be acquired every experiment for optimal correction of fluorochrome spectral overlap. In contrast, FMO controls are cells
that are stained with all the fluorochromes except one fluorochrome. These cells are critical in setting up the cell gating and
522
Gerard Agbayani et al.