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This kit uses fluorescent dye (bisbenzimide,Hoechst 33258) to detect mycoplasma contamination. The dye binds to the A-T-rich region of the DNA, and because mycoplasma DNA has A high content of A-T (55% to 80%), it can be detected by staining it. After staining the cells contaminated by mycoplasma, many uniform fluorescent dots can be seen around the cells, that is, the DNA stain of mycoplasma, indicating that there is mycoplasma pollution. Hoechst 33258 has a maximum excitation wavelength of 346nm and a maximum emission wavelength of 460nm. When combined with double-stranded DNA, Hoechst 33258 has a maximum excitation wavelength of 352nm and a maximum emission wavelength of 461nm.
Operation steps:
Adherent cells:1. The tested cells were inoculated in a sterile 6-well cell culture plate with a inoculation density of 1-2×104. At the same time, the same kind of cells without normal mycoplasma infection were inoculated as negative control.
After 2.5 days, firstly remove the culture medium, then add 1ml fixing solution, and let it stand for 20min,
3. Drain fixing solution and let dry.
4. Add 1ml of Hoechst33258 working liquid (Hoechst33258 working liquid should cover all the tested cells) into each well, and place at 37℃ away from light for 15-20min or at room temperature for 20-30min.
5. Absorb Hoechst 33258 working liquid, add 2ml sterilized ultra-pure water to wash three times, and directly air dry. Air dry, add a drop of sealing liquid, and cover with a cover glass.
6. Fluorescence microscope observation. Ultraviolet excitation light excitation was used to observe whether there were blue fluorescent dots or beaded fluorescent dots around the cells.
Suspended cells:
1. Collect the cells to be detected, 1500rpm, 5min.
2. Smear the collected cells on a slide, add 1ml of fixing solution, and stand for 20min.
3. Drain fixing solution and let dry.
4. Add 1ml of Hoechst33258 working liquid (Hoechst33258 working liquid should cover all tested cells) into each well, and place at 37℃ away from light for 15-20min or at room temperature for 20-30min.
5. Absorb the Hoechst 33258 working liquid, wash the slide with sterile water 3 times, and air dry directly. Air dry, add a drop of sealing solution, and cover with a cover glass.
6. Fluorescence microscope observation. Ultraviolet excitation light excitation was used to observe whether there were blue fluorescent dots or beaded fluorescent dots around the cells.
Result judgment (reference) :
Negative: Only the nucleus of the cell shows yellowish-green fluorescence.
Positive: Except for cells, a large number of uniformly sized fluorescent colored particles can be seen around the cells.
Note:
1.Hoechst working fluid is harmful to human body, please pay attention to protection.
2. Fixed liquid has pungent smell, it is recommended to fix in the fume hood.
3. When detecting mycoplasma, it is best to culture the culture medium without antibiotics for 2 to 3 generations, so that false negative results can be easily avoided.
4. When the kit is used for 6-well plate detection, it can perform 50 detection reactions.
5. To detect mycoplasma contamination, efficient Vero cells of mycoplasma can be used, which can improve the detection sensitivity, and the tested sample will be inoculated into Vero cells for detection.
Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.
