1.Wear appropriate personal protective equipment (PPE), including gloves and a clean lab coat, at all times. It is important to change the lab coat at least once a week.
2.Sttrictly following sterile techniques is vital for safeguarding your cells and preventing contamination. Here are some practical measures:
2.1.Maintain a clean and well-organized cell culture hood to ensure unobstructed airflow.
2.2.Spray with 75% alcohol before placing items in the hood.
2.3.Ensure proper covering of plates and bottles.
2.4.Refrain from swinging your hands and arms over open dishes and flasks.
3.Promptly clean up any spills.
4.Keep the incubator clean and avoid any splashes or spills. Adhere strictly to the cleaning schedule. Regularly disinfect the incubator with bleach. Change or clean the catch-up dish once a week to prevent internal cell contamination.
5.Isolate all new or previously untested cell lines in a designated incubator. Mycoplasmas are particularly prone to spread from one area to another. Whenever possible, don’t place new or questionable cell lines near other cells until they have been tested to ensure they are free from contamination.
6.Cell culture supernatants can be tested or prepared for future use after at least 12 hours of cell culture. Transfer 200 μL of supernatant into a sterile 1.5-mL safe lock tube and incubate the sample at 95℃ for 5 minutes.
7.Prepare the negative control, positive control and samples. Add 1 μL of DNA-free water to the negative control and add 1 μL of the sample to each of the sample tubes and label them. Positive controls were obtained from MycoStripTM. The reaction tubes provided in the kit include the nucleotides, primers and internal control DNA.
8.Prepare the samples for PCR and determine the total volume of Taq DNA polymerase buffer required for the reactions.
9.Following the PCR reaction, use 10 μL of the produced sample, and perform 1.5% agarose gel electrophoresis at 120 V until the electrophoresis exceeds two-thirds of the gel (refer to the example gel in Fig. 1). Use 5 μL of Trans 2 kb DNA ladder to mark the DNA fragments. Store the remaining samples at -20℃ for future use.
10.Following reagent manufacturer’s protocol of Zell Shield®, put 0.5 mL Zell Shield® (100×) to a 45 mL cell medium.
11.When the cells in the 10-cm dish reach 90% confluency, use trypsin to digest the 293 T cells, or directly transfer NK92 and Myla cells into a 15-mL centrifuge tube.
12.Centrifuge the cells at 1,000 rpm for 5 min at room temperature. Remove the supernatant and resuspend the cells in 5 mL 1 × PBS.
13.Carefully discard the supernatant and resuspend the cells with another 5 mL PBS. Gently pipette up and down to mix cells in all plates. Centrifuge all samples for 5 min at 1,000 rpm at room temperature.
14.Wash the cell plates three times with 5 mL 1×PBS as described in eradication step 13 (step4 in Fig. 2).
15.Remove the supernatant and resuspend the cells in fresh medium containing Zell Shield®.
16.According to cell density, repeat steps 12 to 15 (step 2 to 6 in Fig. 2) four times (treatment for a total of 10 days).
17.Collect four supernatants and perform mycoplasma testing as done in PCR procedure.
18.Test the cultures for mycoplasma contamination (refer to the example gel in Fig. 3). Cells cultured without Zell Shield® for an additional 7 days. Afterwards, they should be checked again and then frozen in liquid nitrogen.