Mohler W A, Charlton C A, Blau H M
Stanford University Medical Center, CA, USA.
Biotechniques. 1996 Aug;21(2):260-2, 264, 266. doi: 10.2144/96212st03.
We have developed a spectrophotometric assay for cell number in suspensions of tissue culture cells. For each cell type tested, absorbance between 650 and 800 nm is linearly dependent upon cell density over a 50-fold range and is independent of the color or composition of the medium in which cells are suspended. A standard curve of absorbance vs. cell density is used to estimate cell number with accuracy and reproducibility superior to hemacytometer counting and with speed and ease surpassing use of a Coulter counter. Less than 5000 cells are needed for this quantitation. The same cells that are counted can be maintained live in culture after the reading is taken, thus allowing the growth of cells to be measured within individual cultures over time. The assay should be readily extended to assays of cell number directly within microplate culture wells. The spectrophotometric assay described here is of significant use in all experiments requiring rapid, accurate measurements of cell number, including determinations of cell doubling time and equal plating of parallel cultures.
我们开发了一种用于测定组织培养细胞悬液中细胞数量的分光光度法。对于所测试的每种细胞类型，在650至800nm之间的吸光度在50倍的范围内与细胞密度呈线性相关，并且与细胞悬浮于其中的培养基的颜色或成分无关。吸光度与细胞密度的标准曲线用于估计细胞数量，其准确性和可重复性优于血细胞计数器计数，速度和便捷性超过库尔特计数器的使用。这种定量分析所需的细胞数量少于5000个。计数后的相同细胞在读取数据后可以在培养中保持存活，从而可以随时间测量单个培养物中细胞的生长情况。该测定法应易于扩展到直接在微孔板培养孔内进行细胞数量的测定。这里描述的分光光度法在所有需要快速、准确测量细胞数量的实验中都有重要用途，包括测定细胞倍增时间和平行培养物的等量接种。
