Instructions For UseImportant:
Thaw all components prior to use.Note 1:
Any buffer without sodium azide, serum or protein can be used instead of phosphate buffered saline.Note 2:
For multicolor experiments, the cells can be stained with the antibodies of your choice before or after staining with VivaFix Dye. Antibody staining conditions should be optimized for your assay.
1. Prepare a 500x stock solution by adding 50 μl of dimethyl sulfoxide (DMSO) to a VivaFix Dye vial and mix by vortexing.
2. Wash cells once with phosphate buffered saline, then resuspend cells at 2-3 x 106
cells/ml in phosphate buffered saline. Use 0.5 ml of cell suspension per assay.
3. Add 1 μl of 500x dye stock solution (from step 1) to 0.5 ml cells/assay and mix by vortexing.
4. Incubate the mixture for 30 min at room temperature (RT) or in a 37o
incubator. Protect from light.Note:
The optimal dye concentration and incubation time for different cell lines should be assessed empirically.
5. Wash cells twice in phosphate buffered saline.
6. Fix cells as desired. For 3.7% formaldehyde:
-Suspend cells in 900 μl of phosphate buffered saline following step 5
-Add 100 μl of 37% of formaldehyde to the cell suspension
-Incubate for 15 min at room temperature, then wash cells twice with phosphate buffered saline
7. Resuspend cells in the appropriate flow analysis buffer.
8. Sort cells with an S3e Cell Sorter™
, analyze cells with a flow cytometer, view and capture an image of cells with the ZOE™ Fluorescent Cell Imager
, or view cells with a conventional fluorescence microscope.