Fluorescence detection of Mycoplasma
Fluorescence detection of Mycoplasma
Cells were cultured for at least one week in the absence of antibiotics, culture supernatants were transferred to the indicated cells in early logarithmic growth, incubated for 3 to 5d , and the cells were fixed and stained to look for fluorescence outside the nucleus.
Operation method
Program 19.2 Fluorescence Detection of Mycoplasma
Principle
Cells were cultured for at least one week in the absence of antibiotics, culture supernatants were transferred to the indicated cells in early logarithmic growth, incubated for 3 to 5d , and the cells were fixed and stained to look for fluorescence outside the nucleus.
Materials and Instruments
Antibiotic-free cell culture supernatant from 7-day monolayer culture or after centrifugation Indicator cells Move 1. Inoculate the indicator cells (e.g. 3T6, NRK, Vero, A549) in a culture dish (6 cm diameter) without antibiotics, the number of inoculated cells can be sufficient to produce a 50%~60% confluent layer in 4~5 days (e.g. 2×104 NRK or 1×105 A549 in 5 ml of culture medium). Caveat If the culture reaches full confluence before the end of the assay, staining will be inhibited and the mycoplasma will be difficult to visualize. For more product details, please visit Aladdin Scientific website.
Hoechst 33258 dye BSS-PR D-PBSA deionized water fixative sealer
Petri dishes
2. Add 1.5 ml of culture medium from the culture being tested.
3. Incubate the above cultures until the cells reach 50%-60% confluence.
4. Remove the culture solution and discard it.
5. Rinse the monolayer with BSS-PR (Hanks' BSS without phenol red) or D-PBSA (Dulbecco's PBSA without Ca2+, Mg2+ ) and discard the rinse solution.
6. Add fresh BSS-PR or D-PBSA diluted 50:50 in fixative (freshly prepared glacial acetic acid: methanol (1:3 on ice)), rinse the monolayer and discard the rinse solution.
7. Add pure fixative, rinse, discard rinse solution.
8. Add more fixative (approx. 0.5 ml/cm2 ) and fix for 10 min.
9. Remove the fixative and discard the fixative.
10. If preparing for storage, dry the monolayer culture completely (specimens can be accumulated at this stage and stained later).
11. if you are going to stain directly, rinse with deionized water and discard the rinse solution.
12. Add Hoechet 33258 (Hoechst 33258 dye, 50 ng/ml dissolved in BSS (BSS-PR) or D-PBSA without added phenol red) to the BSS-PR or D-PBSA solution and stain for 10 min at room temperature.
13. Remove the staining solution and discard it.
14. Rinse the monolayer with water and discard the rinse solution.
15. Seal the coverslip with a drop of sealer (buffered glycerol solution) and wipe away excess sealer from the edge of the coverslip.
16. Fluoresce the monolayer with a 330/380 nm laser filter and an LP 440 nm barrier filter.