XTT method steps for cell activity detection

In addition to CCK8 and MTT, there is also an XTT detection method for cell activity detection!

The XTT method for cell viability detection is a method for detecting cell proliferation and activity. The detection principle is that XTT is a tetrazolium nitrogen derivative similar to MTT, which can be reduced by live cell mitochondrial dehydrogenase to a water-soluble brown carbamate product. When XTT is used in conjunction with electronic coupling, the production of methylcellulose is positively correlated with the degree of cell proliferation.

In the cell activity detection experiment, XTT reagent and electron coupling reagent are mixed in a certain proportion, added to the tested cells, and then incubated. During the incubation process, living cells will reduce XTT reagents to methylcellulose products, while dead cells cannot undergo this reduction reaction. By detecting the production of methylcellulose products, the degree of cell proliferation and cell viability can be determined.

The use of XTT method for cell activity detection has the advantages of simple operation, high sensitivity, and good repeatability. Meanwhile, this method has low cytotoxicity and is suitable for detecting the activity of various mammalian cells.

Reagents and materials:

(1) XTT: Prepare 6.6mmol/L of preheated culture medium at 60 ℃, filter and remove bacteria, and prepare it for use immediately;

(2) Phenazine dimethyl sulfate (PMS): prepared with PBS to 220mmol/L, stored at 4 ℃ in dark for 20 days;

(3) XTT/PMS: Mix XTT and PMS in a 1:1 ratio (when in use).

XTT method operation steps:

(1) Stimulate the proliferation reaction, mix XTT reagent and electron coupling reagent in a certain proportion, add them to the cell culture medium, and continue to cultivate for a certain period of time;

(2) Add XTT/PMS 20 to each well 2 hours before termination of cultivation μ l. Mix well and then cultivate for 2 hours;

(3) Measure the absorbance at 450nm on the enzyme-linked immunosorbent assay, with a reference wavelength of 655nm.

Result Analysis

Calculate SI: SI=mean OD of test hole/mean OD of control hole

Precautions for XTT method operation:

(1) The quality and activity of mitogen are key factors in determining lymphocyte proliferation response. The quality and activity of products from different manufacturers vary, and the optimal stimulation amount used varies. Therefore, before conducting experiments, the optimal stimulation amount should be determined in advance.

(2) Detect T cell proliferation response using PHA or ConA for mitogen; B cell proliferation response using LPS or SPA; T. The B cell proliferation reaction is treated with PWM.

(3) The proliferative cells should maintain high activity, generally ≥ 95%. The reaction cells prepared by monoclonal antibody separation can inhibit cell proliferation due to the direct action of preservatives (such as NaN3) or antibodies. In addition, high or low concentrations of proliferating cells are not conducive to cell growth.

(4) Cell culture medium should be sterile and free from mycoplasma contamination, especially calf serum should be heated to inactivate complement to avoid contamination by LPS or other substances that can promote cell proliferation. Therefore, low background calf serum should be selected before the experiment. Human "AB" type serum or autologous serum should also be heated to inactivate complement.