CyTRAK Orange™ example protocols
LIVE CELL STAINING WITH CyTRAK OrangeTM FOR NUCLEUS AND CYTOPLASM DISCRIMINATION
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LIVE CELL STAINING WITH CyTRAK OrangeTM FOR NUCLEUS AND CYTOPLASM DISCRIMINATION
Appropriate for:
- Wide-field camera-based imaging
- Confocal Laser Scanning Microscopy
Reagents required:-
- CyTRAK OrangeTM
- Phosphate Buffered Saline (PBS, without sodium azide) or other culture medium
CyTRAK OrangeTM is intended for research purposes only
- 1. Read the supplied Material Safety Data Sheet before handling CyTRAK OrangeTM
- 2. Since no washing step is required, CyTRAK OrangeTM will usually be the final staining procedure, after any cell treatment or labelling, prior to analysis.
- 3. Prepare cells for staining with CyTRAK OrangeTM. Resuspend cells in appropriate buffer such as PBS at a concentration of ≤4 x 105 / ml in a test tube. For adherent cells estimate the number of cells based on confluence level or tissue section dimensions.
- 4. Add CyTRAK OrangeTM directly as supplied following the 10 µM or 20 µM pipetting volumes in table 1 below. This will be as an overlay for adherent cells / tissue sections, added to the chamber/well liquid directly or in fresh medium following a wash step.
- 5. Gently mix and then incubate for 20-60 minutes at room temperature. NOTE: Protect from the light during this incubation period if other (immuno-) fluorescent stains have been applied to the cells, prior to the CyTRAK OrangeTM labelling, and which may otherwise suffer photo-bleaching. CyTRAK OrangeTM staining is accelerated at 37ºC and maybe reduced to 10-15 min. CYTRAK OrangeTM stains intact, live, fixed, permeabilized and dead cells.
- 6. Cells can be analysed directly without further treatment or washing. NOTE: To maintain the capacity in the assay for cell compartment discrimination we do not recommend a further washing protocol. If washing is necessary then the cytoplasmic signal will be removed, however the nuclear signal is well maintained.
Considerations:
It is important to consider the combinations of fluorochromes and filters for the experiment:
EXCITATION: CyTRAK OrangeTM may be excited by a wavelength range from 488 nm up to 540 nm (Exλmax 520 nm) (see excitation spectra for CyTRAK OrangeTM).
- 488 nm excitation offers the most simple and optimal wavelength additionally it is available on most imaging instruments.
- NOTE: The key benefit of CyTRAK OrangeTM is that is can be co-excited with eGFP or eYFP, and the emission profile enables it to be robustly separated from either of these fluorescent constructs.
EMISSION: this starts at 580 nm (Emλmax 615 nm intercalated to dsDNA). See emission spectra for CyTRAK OrangeTM
- Suitable filters include 590LP; or a 630/60 nm band-pass or 615/50 nm band-pass for imaging with eGFP or eYFP constructs
See table 2 below for recommended filter combinations for imaging.
Table 1:
Ready reckoner for volumes of CyTRAK OrangeTM (5mM) required for various cell concentrations:-
Cell sample preparation: | Volume of CyTRAK OrangeTM (as supplied) required for a concentration of: | |||
No. of cells: | in volume: | 5 µM | 10 µM | 20 µM |
1 x 106 | 2500 µl | 2.5 µl | 5 µl | 10 µl |
4 x 105 | 1000 µl | 1 µl | 2 µl | 4 µl |
2 x 105 | 500 µl | 0.5 µl | 1 µl | 2 µl |
1 x 105 | 250 µl | 0.25 µl | 0.5 µl | 1 µl |
5 x 104 | 125 µl | 0.13 µl | 0.25 µl | 0.5 µl |
Table 2:
Typical filter combinations for CyTRAK OrangeTM
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