TY - JOUR
T1 - A fluorometric approach to the quantitation of cell number with application to a cell adhesion assay
AU - Lewinsohn, David M.
AU - Nickoloff, Brian J.
AU - Butcher, Eugene C.
N1 - Funding Information:
This work was supported by National Institutes of Health Grants AI19957 and AM53590. D.M.L. is a predoctoral fellow of the Cancer Biology Program, supported by United States Public Health Service training Grant 5T32 CA 09302-09. E.C.B. is an Established Investigator of the American Heart Association. We would like to thank Philip Streeter and Deborah Hardy for their critical reading of the manuscript, and Marsha Smullens for excellent technical assistance.
PY - 1988/5/25
Y1 - 1988/5/25
N2 - We describe a fluorometric approach to the determination of cell number. Cells are covalently labeled with fluorescein isothiocyanate (FITC) under physiologic conditions. Cells are lysed with detergent, and released fluorochrome is assessed quantitatively with a fluorescence spectrophotometer. Fluorescence varies linearly with regard to cell number over a wide range of concentrations, and allows detection of as few as 8 × 103 cells/ml. We describe the effect of different time and temperature incubations of FITC-labeled cells on fluorescence intensity, and we use the method to analyze the binding of peripheral blood mononuclear leukocytes to interferon-gamma-treated keratinocytes. Finally, we demonstrate that the results in an adhesion assay are comparable to those obtained with 51Cr-labeled cells. Quantitative fluorescence analysis of cell number offers a safe, inexpensive, rapid, and accurate method of determining cell number without the biological hazard and waste disposal problems associated with radioactive labeling.
AB - We describe a fluorometric approach to the determination of cell number. Cells are covalently labeled with fluorescein isothiocyanate (FITC) under physiologic conditions. Cells are lysed with detergent, and released fluorochrome is assessed quantitatively with a fluorescence spectrophotometer. Fluorescence varies linearly with regard to cell number over a wide range of concentrations, and allows detection of as few as 8 × 103 cells/ml. We describe the effect of different time and temperature incubations of FITC-labeled cells on fluorescence intensity, and we use the method to analyze the binding of peripheral blood mononuclear leukocytes to interferon-gamma-treated keratinocytes. Finally, we demonstrate that the results in an adhesion assay are comparable to those obtained with 51Cr-labeled cells. Quantitative fluorescence analysis of cell number offers a safe, inexpensive, rapid, and accurate method of determining cell number without the biological hazard and waste disposal problems associated with radioactive labeling.
KW - Cell adhesion
KW - Cell number
KW - Fluorescein isothiocyanate
KW - Fluorescence spectroscopy
UR - http://www.scopus.com/inward/record.url?scp=0023883247&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023883247&partnerID=8YFLogxK
U2 - 10.1016/0022-1759(88)90087-7
DO - 10.1016/0022-1759(88)90087-7
M3 - Article
C2 - 3131438
AN - SCOPUS:0023883247
SN - 0022-1759
VL - 110
SP - 93
EP - 100
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1
ER -