Rapid, one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry

Journal Article · Fri Jan 01 00:00:00 EST 1982 · Cytometry (Baltimore); (United States)

DOI:https://doi.org/10.1002/cyto.990030204· OSTI ID:5466688

Crissman, H A; Steinkamp, J A

Detailed, simplified techniques are described for simultaneous staining and analysis of DNA and protein in a number of mammalian cell types. Cell staining involves the addition of appropriate dye mixtures to unfixed or ethanol-fixed cells and subsequent analysis of cell populations in the staining reagents generally within 10 to 20 minutes. The approach is novel in that no centrifugation steps are involved during the staining procedure, thus, eliminating cell clumping and cell loss and making the procedures appropriate for samples containing limited numbers of cells. For single wavelength analysis, staining of DNA and protein in ethanol-fixed cells was accomplished with a dye solution containing propidium iodide, fluorescein isothimocyanate and RNase. After 20 minutes at room temperature cells were analyzed using the 488 nanometer (nm) laser excitation line. For dual laser analysis the following dye combinations were employed without RNase: mithramycin-rhodamine 640, mithramycin-substituted rhodamine isothiocyanate, Hoechst 33342-rhodamine 640 and Hoechst 33342-rhodamine isothiocyanate. Unfixed cells were also stained with the Hoechst 33342-rhodamine 640 dye combination. Mithramycin was excited at 457.9 nm, Hoechst 33342 at 333-363 nm, and rhodamine dyes at 568 nm. Cell types analyzed included Chinese hamster ovary cells, cultured mouse colon 26 cells, mouse embryo forelimb bud cells, and rat cell obtained by lung lavage.

• Research Organization: Los Alamos National Lab., NM
• OSTI ID: 5466688
• Journal Information: Cytometry (Baltimore); (United States), Vol. 3:2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA
STAINS
EMBRYONIC CELLS
CYTOLOGICAL TECHNIQUES
CELL CULTURES
HAMSTERS
LARGE INTESTINE
LASER RADIATION
LASERS
MICE
OVARIES
PROTEINS
RATS
STIMULATED EMISSION
ANIMAL CELLS
ANIMALS
BODY
DIGESTIVE SYSTEM
ELECTROMAGNETIC RADIATION
EMISSION
ENERGY-LEVEL TRANSITIONS
FEMALE GENITALS
GASTROINTESTINAL TRACT
GONADS
INTESTINES
MAMMALS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANS
RADIATIONS
RODENTS
VERTEBRATES
550300* - Cytology