Mammalian cell in full culture medium staining methods[2]:
(1) Cell planking: Digestive separation of cells and resuspend in complete medium to a concentration of 2-4 × 105 cells/ml.
Note: Attached cell cultures (e.g., coverslip cultures or chambered wells) can be stained in a 1-2-ml staining volume overlayering a 4-cm2 surface area.
(2) Prepare staining solution: Add 4 µl of 5 mM DRAQ5 acidified stock per ml culture medium (20 µM final).
Note: Nuclear discrimination is achievable at 2.5 to 5 µM, and it is unlikely that concentrations >30 µM would be required.
(3) Fluorescence staining: Incubate 5 to 15 min at 37°C.
Note: Overstaining cannot occur.
(4)Wash (optional): Centrifuge cells 3 to 5 min at 800 × g, 37°C. Discard supernatant and resuspend in complete medium with 10 mM HEPES at 4 × 105 cells/ml.
(5) For flow cytometry: Use conventional pulse analysis for doublet discrimination and analyze parameters using appropriate software.
(6) For laser scanning microscopy: Collect fluorescence images using a 695 nm long-pass filter.
Fixed cells staining methods[2]:
(1) Fixed cells:Use 4% paraformaldehyde in PBS for 30 min with resuspension in an aqueous buffer (e.g., PBS).
(2) Fluorescence staining: similar concentrations of dye and similar incubation conditions can be used as for live cells.
References:
[1]. Smith PJ, et al. A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry. J Immunol Methods. 1999 Oct 29;229(1-2):131-9.
[2]. Smith PJ, et al. DRAQ5 labeling of nuclear DNA in live and fixed cells. Curr Protoc Cytom. 2004 May;Chapter 7:Unit 7.25.