Department of Biochemistry, The University of Kansas, Lawrence, KS, 66045, U.S.A..
Cytotechnology. 2000 Jan;32(1):63-75. doi: 10.1023/A:1008121125755.
A method is described for estimating the numbers ofanimal cells in multi-well culture by simultaneouslymeasuring the lactate dehydrogenase activity of thetotal culture and the medium. The difference betweenthe two reflects the dehydrogenase content of thecells and correlates with cell number. This LDH/INTmethod was tested using several lines of normal andtransformed suspension and adherent cells. Thelactate dehydrogenase activities of duplicate cultureswere determined colourimetrically using reactioncocktails containing lactate, NAD(+), diaphorase,and p-iodonitrotetrazolium violet, with and withoutTriton X-100. The difference in absorbance at 490 nm(DeltaA(490) = A(490, test) - A(490, control)) was used to calculate the lactatedehydrogenase activity of the total culture (+ Triton)and the medium (- Triton). The cellular lactatedehydrogenase activity (difference between totaland medium dehydrogenaseactivities) was proportional to viable cell number. The effects on cell growth of four metabolicinhibitors, sodium azide, actinomycin D,cycloheximide, and taxol, were determined using theLDH/INT assay and direct cell counting. The inhibitorconcentrations that caused decreases in the LDHactivity and cell number by 50% were similar. TheLDH/INT assay is quick and sensitive, works equallywell for adherent and suspension cells, and providesinformation about LDH activities of both the mediumand cells. It is particularly useful for screeningpotential cell-growth inhibitors.
一种通过同时测量总培养物和培养基中的乳酸脱氢酶活性来估算多孔培养物中动物细胞数量的方法。两者之间的差异反映了细胞的脱氢酶含量，并与细胞数量相关。该 LDH/INT 方法已用于测试几种正常和转化的悬浮和贴壁细胞系。使用含有乳酸、NAD(+)、二氢叶酸还原酶和 p-碘硝基四唑紫的反应混合物，通过比色法测定重复培养物的乳酸脱氢酶活性，有和没有 Triton X-100。490nm 处吸光度的差异（DeltaA(490) = A(490，测试) - A(490，对照)）用于计算总培养物（+Triton）和培养基（-Triton）的乳酸脱氢酶活性。细胞内乳酸脱氢酶活性（总酶和培养基酶活性之间的差异）与活细胞数成正比。使用 LDH/INT 测定法和直接细胞计数法测定了四种代谢抑制剂（叠氮化钠、放线菌素 D、环己酰亚胺和紫杉醇）对细胞生长的影响。导致 LDH 活性和细胞数降低 50%的抑制剂浓度相似。LDH/INT 测定法快速灵敏，对贴壁和悬浮细胞同样有效，并提供了培养基和细胞中 LDH 活性的信息。它特别适用于筛选潜在的细胞生长抑制剂。
