Direct measurement of cell numbers in microtitre plate cultures using the fluorescent dye SYBR green I.
The quantitation of cell numbers is essential for many experimental procedures. This study describes the use of the fluorescent DNA binding dye, SYBR green I, to quantitate accurately cell numbers in the wells of microtitre plates by determination of DNA content. The assay involves a single-step procedure and is sensitive to as few as 1000 cells/well. The advantages of SYBR green I are the low level of background fluorescence in the absence of DNA and the similarities of its absorption and emission spectra to those of fluorescein, which makes it possible to use a wide range of equipment for detection of the amount of bound dye. A particular application of the method is the normalisation of data generated by enzyme-linked immunosorbent assays on whole cells. The procedure is applicable to both fixed and unfixed cells, although for fixed cells permeabilisation provides for greater sensitivity.
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. mark.myers@med.monash.edu.au
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D000818:Animals; D052160:Benzothiazoles; D019556:COS Cells / Q000166:cytology; D002452:Cell Count / Q000379:methods*; D002478:Cells, Cultured / Q000166:cytology; D003959:Diamines; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D007328:Insulin / Q000032:analysis; D009930:Organic Chemicals; D011384:Proinsulin / Q000032:analysis; D011804:Quinolines; D013997:Time Factors
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D007328:Insulin; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D011384:Proinsulin