Core Services | hESC/iPSC Core | hESC Laboratory | Stem Cell Core Lab Rules

Stem Cell Core Lab Rules

Mycoplasma Testing:

  1. Any cells to be transferred into the hESC Core Lab incubators must be tested for mycoplasma contamination. Testing for mycoplasma must be conducted by the Stem Cell Core Lab or the Yale Virology Lab (Brady Memorial Labs, Rm. B33). The Core Lab testing fee is $31/sample. The Virology Lab fee is $40/sample. When the PCR assay gives borderline results (PCR becomes positive at a cycle number over 35 cycles and there is a clear band when run on a DNA gel), retesting will be performed.
  2. For frozen cells, we can test one vial of a particular lot. If you only have one vial, you will need to thaw and expand the cells in your lab prior to mycoplasma testing. After the cells test negative for mycoplasma, you can move them into the core lab.
  3. Any cells that exited the hESC Core laboratory and then need to reenter the hESC Core Lab incubators must be retested for mycoplasma contamination before reentering.
  4. Anyone using the hESC Core lab incubators must submit cells on the first Monday of each month for mycoplasma testing. Testing will be performed in the first week of each month. Users can batch their cell lines together for screening. If the testing is positive for mycoplasma, each cell line will then be tested individually. We will need to conduct the testing on individual clones as soon as possible after the initial positive result. If we are too busy, we may ask you to send the testing to the Virology lab. If you cannot submit ~100,000 cells for testing on the first testing date, an alternative testing date will be scheduled for you, but there may be a delay that could slow down your experiments.
  5. Frozen cells from July – Nov 2012 need to be tested for possible mycoplasma contamination. The hESC Core will perform the testing ($31/sample) or this can be done in the Virology lab.

Cell Culture Room

  1. Please make sure to step on the sticky mat as you enter the cell culture room.
  2. Wear disposable lab coat and gloves inside the cell culture room at all times. When exiting the lab, please take off the coat and discard gloves before touching the door handle.
  3. Empty liquid waste right after you are done with your work and add 50ml bleach to the waste bottle in preparation for the next user.
  4. Transfer solid biohazard waste box to the biohazard storage room and set up a new one once the bin is full.

Flow Hoods and Scraping Stations:

  1. Use your own pipette tips, pipettes, plates, media, tubes, 15/50ml conical tubes and gloves…
  2. Clean the hood before and after your work with Bacdown and 70% ethanol. Spray all of the materials that you bring into the hood with ethanol. Try not to block the air flow in the back or front of the flow hood with pipette boxes, supplies, your arm, etc. Remove everything from the flow hood once your work is completed. Please leave the flow hood on and running continuously.
  3. Use your own scraping tools in scraping stations (Autoclaved glass pipettes or sterile pipette tips work well). Please empty the hood once you are done. Please remember to turn on the scraping hood UV light after each scraping session.
  4. Hood 3 is assigned for virus / BSL2 work. If you are working with BSL2, please book hood 3. Remember to double glove when working with BSL2 materials and label all BSL2 equipment and storage boxes.
  5. Try to be as gentle as you can while you are working inside the cell culture room. Limit your talk while scraping.

Incubators:

  1. Write your name on the incubator sign-up sheet to reserve your space.
  2. Label your plates and dishes clearly with your name and date. If plates are not clearly labeled, they will be discarded.
  3. Let Jason or Yinghong know immediately if you notice any cells within the incubator that are contaminated. Contaminated plates must be discarded immediately.
  4. Be careful while working in the incubator to avoid touching the neighboring plates.
  5. Clean any spills in the incubator right away with 10% Bacdown and 70% ethanol and wait for it to be dried before putting your plate back and closing the door.

Medium and Reagent Ordering:

  1. Media are prepared on Tuesday and Friday afternoons. Orders for media must be placed on the media order sheet at least one day before they are prepared. Prepared media will be placed on the 2nd shelf of the Stem Cell Core refrigerator for pick up.
  2. If you order supplies (i.e. Matrigel, Dispase, Accutase, etc.), please fill out the supplies order sheet (complete with your charging PTAEO) before removing material from the lab. If you notice that supplies or reagents are low in number, please let Jason or Yinghong know immediately.
  3. In response to requests from some users, we are looking into what the cost recovery charge would be for the hESC Core Laboratory to provide plates of normal hESC on a regular basis. Please call if you have questions about this service.