Mycoplasma Assay Protocol

Introduction

Note: This protocol is adapted from the MycoAlert Protocol as detailed here, but for use in the Lai Lab at UCLA.

This assay is used to test cell culture media for the presence of Mycoplasma, a genus of bacteria that can contaminate cell samples.

Required Reagents

Before we begin, you will need to ensure that you have access to the following reagents and equipment:

Reagents

Item	Location
Lucetta 2 Luminometer	For our purposes, this is found in the Nathanson Lab at CHS 23-376, although you will likely need someone from the Nathanson Lab main room to let you in.
Secondary carrying container for bioharzardous materials	You can borrow one from the Nathanson Lab, or use your own. Both will need to be sterilized with 70% isopropyl alcohol after use.
Cell culture media	Varies.
96 Well microtiter plate	Bring your own, or use the one in the Nathanson Lab for ongoing bioassays.
100 µL pipette	Lab Benchtop.
100 µL pipette tips	Lab Benchtop.
100 µL of UltraPure H2O (or buffer) for a control sample	Lab Benchtop.
MycoAlert Substrate	In MycoAlert Kit, Cat #: LT07-318. Stored in the Lai lab in the -80ºC freezer as 310 µL aliquots, which is good for 3 samples. Note that aliquots should not be freeze/thawed, so discard any excess once you’re finished.
MycoAlert Reagent	In MycoAlert Kit, Cat #: LT07-318. Stored in the Lai lab in the -80ºC freezer as 310 µL aliquots, which is good for 3 samples. Note that aliquots should not be freeze/thawed, so discard any excess once you’re finished.

Protocol

Preparing the Assay

1. Head over to the Nathanson Lab main room. It is located at CHS 23-234. You should contact someone ahead of time. Otherwise, any Lucetta 2 Luminometer will do.
2. Next, take out a microtiter plate designated for biohazardous samples. The Nathanson Lab usually keeps a few in use in the middle drawer on the first lab bench once you enter the room. These microtiter plates are often used for assays involving mouse blood, so treat them as biohazardous. You should do the same if you use your own plate.
3. In the microtiter plate, place 100 µL of sample (or control) into unused wells.
4. Place the plate in your biohazardous secondary container for transport.
5. Go to the assay room at CHS 23-376. A member of the Nathanson Lab will likely need to accompany you to let you in and log you into the machine.
6. To each sample, add 100 µL of thawed MycoAlert Reagent (labeled “R” on aliquots) and wait 5 minutes.

Running the Assay

1. Boot up the CLARIOstar program.
2. Select Manage Protocols and then click on Myco Test.
3. In the window, click on the Layout tab and Empty out all cell wells. You can do this by selecting the entire plate and clicking on the Empty button.
4. Click Sample and select the wells on the plate that contain your samples (including the control). Click Start Measurement.
5. In the second window that pops up, uncheck Gain Adjustment.
6. Once your 5 minutes are up, push the small black button above the myco tray insert on the machine to open it. Place your microtiter plate into the tray.
7. On the new window in CLARIOstar, double check that Gain Adjustment is unchecked. Then, hit Start Measurement when you’re ready.
8. Once the program is finished, write down the values that pop up for each sample. These values are your first measurement (A).
9. Remove the microtiter plate from the machine.
10. Next, add 100 µL of thawed MycoAlert Substrate (labeled “S” on aliquots) to each sample. Wait 10 minutes.
11. Repeat steps 2-5. Ensure that Gain Adjustment is unchecked, and then run again.
12. Once the program is finished, write down the values that pop up for each sample. These values are your second measurement (B).
13. Close the application and log off the computer (if in the Nathanson Lab, leave it on). Return the microtiter plate to where you found it, if in the Nathanson Lab (no need to aspirate the samples), otherwise dispose of the media as if it were biohazardous. Sterilize your biohazardous secondary container with 70% isopropyl alcohol.

Next, calculate the following ratio for each of your samples, where B is your reading after adding MycoAlert Substrate and A is your reading after adding MycoAlert Reagent. Use the table below to determine the outcome of the assay.

MycoAlert Result: Ratio = B/A

Ratio Value	Result from the MycoAlert Assay
Less than 1.0	Negative for mycoplasma.
1.0-1.2	Inconclusive. Quarantine the cells for 24-48 hours and retest.
Greater than 1.2	Positive for mycoplasma.