BALB/c thymocytes were uncultured (left) or cultured overnight at 37°C (right) and then stained with 1 ĩL of Fixable Viability Dye eFluorŪ 450 per ml of thymocytes that were resuspended at 5x106 cells/ml in PBS. Total cells were used for analysis.
Contents: Fixable Viability Dye eFluorŪ 450 Catalog Number: 65-0863 Formulation: Dye in DMSO, pre-diluted to test size Storage Conditions: Store at less than or equal to -70°C. Protect from light and moisture.
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65-0863-14
100 tests
65-0863-18
500 tests (5 X 100 tests)
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Flow Cytometry Product Notes: Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µL to 5 µL. Please check your antibody vial for the recommended test size. Fluorochrome Replacements: eBioscience is in the process of replacing all Alexa FluorŪ 647 conjugated products with eFluorŪ 660 conjugated products.
Description
Fixable Viability Dye eFluor® 450 is a viability dye that can be used to irreversibly label dead cells prior to cryopreservation, fixation and/or permeabilization procedures. Unlike 7-AAD and propidium iodide, cells labeled with Fixable Viability Dyes can be washed, fixed, permabilized, and stained for intracellular antigens without any loss of staining intensity of the dead cells. Thus, using Fixable Viability Dyes allows dead cells to be excluded from analysis when intracellular targets are being studied. Fixable Viability Dyes may be used to label cells from all species.
Fixable Viability Dye eFluor® 450 can be excited by the violet (405 nm) laser line and can be detected using standard filter sets for eFluor® 450 or Pacific Blue® (450/50 band pass filter). Please make sure that your instrument is capable of detecting this dye. For compensation, it is recommended to use a sample of the cells of interest stained with the Fixable Viability Dye. If the percentage of dead cells is expected to be less than 5%, then it is recommended to take a small aliquot of cells and heat them at 65°C for 1 minute then immediately place on ice for 1 minute. After this treatment, the heat-killed cells can be combined 1:1 with live cells and then stained with the Fixable Viability Dye.
Fixable Viability Dye eFluor® 450 is supplied as a pre-diluted solution prepared in high-quality, anhydrous DMSO. It should be protected from light and moisture. Store at -80°C with dessicant. It may be freeze-thawed up to 20 times. Allow vial to equilibrate to room temperature before opening.
Applications Reported
Fixable Viability Dye eFluorŪ 450 has been reported for use in flow cytometric analysis.
Applications Tested
Fixable Viability Dye eFluorŪ 450 has been pre-titrated and tested by flow cytometric analysis of mouse thymocytes. Fixable Viability Dyes are fully compatible with both IC Fixation and Permeabilization Buffers and the Foxp3 Buffer Set. This can be used at 1 μl/ml of cells resuspended at 1-10x106 cells per ml in azide-free and serum/protein-free PBS. It is recommended that the concentration used be determined by each investigator for optimal performance in the assay of interest.
Special Notes
Staining with Fixable Viability Dye eFluor® 450 may be done before or after surface staining. Cells may be cyropreserved after staining with Fixable Viability Dye eFluor® 450 with no adverse effect on staining intensity of dead cells after thawing.
Protocol
To view protocol, please download the TDS by clicking the PDF button above.