LaCava Research Wiki

An easy test to confirm the existence of mycoplasma in your cell culture.

Material:

• Put microscope coverslip (18mmx18mm) in the bottom of your dish and leave 1X DPBS for a couple of minutes to make sure coverslip is not moving. Aspire 1X DPBS.
• Cells are cultured on a dish with coverslip(1 dish/cell type to test).
• Cell confluence does not represent an issue, since you want to check the presence or absence of mycoplasma contaminants in the cytoplasm.
• 4% PFA solution
• 1X DPBS
• DAPI soltion ( diluted from stock solution as recommended by manufacturer)
• Mounting media

Protocol:

• Remove cell culture medium and incubate cells for 15 minutes with 4% PFA at room temperature.
• Wash the cells twice with 1X DPBS.
• Incubate cells with DAPI solution for a minutes.
• Wash the cells 2-3 times with 1X DPBS.
• Aspire remaing DPBS carefully ( not touch the glass cover in the bottom of dish)
• Leave couple of drop mounting media on microscope glass and put the coverslip facing down on the glass. The mounting media should cover the surface of coverslip.
• Leave the microscope glass in the dark place four 6-12 hours to dry.
• Look at the fixed cells with a classic epifluorescent microscope. In normal cells we expect to see no emission outside of nucleus.

Following images were obtain from NTERA2/D1 and N2102EP/2A6 in order:

NTERA2/D1

N2102EP/2A6