Cell Culture Training (freezing and thawing)

NOTE: Access to UC Berkeley facilities and services requires university approval which takes as little as fast as 24 hours. First time customers on Second Lab will be prompted to complete approval requirements during checkout. We encourage you to book your 15 minute onboarding with the Second Lab team through the following link: Click here to schedule your onboarding (15 mins)!

During checkout, you will be presented with a form for the respective service you're requesting. Additional requirements may be applicable if your service requires physical access to University of California facilities.

Frequently Asked Questions

Q: When is the latest I should place my order for: 

An adherent line with a desired confluence?

A:  9am the day before you would like to be ready. Please note we only keep a certain number of plates per cell line. For larger orders (i.e. more than 1-2x10cmTCD or more than 1x15cmTCD), try to place orders at least 3 days before your desired pick up date. Additionally, if we are not currently growing the cell line, we may need additional time to make your order. We will let you know of any delays in advance. 

An adherent line with a desired cell count?

A: Before 9am the same day you would like it to be ready. Please note that if the cell count is higher than the amount we have in stock or if we are not currently growing the cell line, we will let you know in advance. 

An insect cell suspension line? 

A: HIGH5 (split M, W, F): by 9am on the split day you would like it to be ready. For orders greater than the equivalent of 1x1L @ 1e6, please place your order by 9am the split day prior to your pick up date (e.g. A 2L HIGH5 order with a density of 1e6/ml for pick up on a Wednesday should be placed by 9am the Monday before. A Monday pick up for the same order should be placed by 9am the Friday before). 

SF9 (split M, W, F): by 9am on the split day you would like it to be ready. For orders greater than the equivalent of 2x1L @ 1e6, please place your order by 9am the splitting day prior to your pick up date (Assuming no larger orders are in front of your order).

EXPI SF9 (split M, Th): by 9am on the split day prior to your pick up date. For orders greater than the equivalent of 100ml @ 2e6, please place your order by 9am at least TWO splitting days prior to your pick up date. For orders greater than the equivalent of 1.5L @ 1e6, please place your order at least FOUR splitting days (~2 weeks) before your pick up date. 

*for pick up dates that are not on a split day (e.g. Tues), you should place orders as if they are to be picked up on the split day prior (e.g. A HIGH5 order <1L @1e6/ml for pick up Tuesday should be placed Monday by 9am. A HIGH5 order >1L @ 1e6/ml for pick up Tuesday should be placed Friday by 9am.)

A mammalian suspension line? 

293 GNTI (split M,Th): by 9am the splitting day prior to your pick up date

EXPI F, EXPI GNTI: (split M, Th): by 9am the splitting day prior to your pick up date

*Please note larger orders (293 GNTI: >100ml @ 1e6, EXPI F: >125ml @ 1e6) will take additional time. These orders should be placed at least a week in advance. But we suggest at least 2 weeks especially for 293 GNTI orders larger than the equivalent of 2L @ 1e6/ml)

Q: Do I need to do anything after I place my order?

A: If you plan to use recycled flasks for your suspension lines, please bring them at least two splitting days in advance. We do not keep black well plates. If you request them for an order, you must provide your own a day in advance. 

Q: When will my order be ready?

A: You can expect your order to be ready on your desired pick up date. For same day orders, pick up is after 2pm. If there is a delay, we will inform you ahead of time. For mycoplasma and testing and STR validation, please allow up to a week before results. 

Q: Will you contact me when my order is ready? 

A: No. Once you place your order, expect that it will be ready by 2pm on the day you requested. The only exceptions are suspension lines that require us to thaw the same day. Those will be ready by 4:30pm at the latest. We will only contact you if there is a reason for delay or need to clarify a request. 

Q: When will I receive my shipped order?

A: Next day. We provide overnight shipping Monday-Thursday (we don't ship on Friday). As long as we have your lab's chartstring, FedEx account number, and shipping information, orders placed before 11:30am will be received the next day. If there's an issue or delay, we will let you know ahead of time.

Q: What passage is my cell line?

A: Cell line passages can be found in the upper left corner of your dish. It will be marked with a “P” followed by the passage number. 

Q: What passage is my frozen cell line?

A: You can request that information when you place your order and we will let you know when you come by to pick it up. 

Q: How do I pick up my order?

A: Find the pick up shelf (insect cells are in the insect cell incubator next to Freezer 1 or in small order incubator located on the counter across from the insect cell incubator on the right side of the large centrifuge. Mammalian suspension lines are in incubator 1 or 2, adherent lines are in incubator 3). Put on gloves and spray with ethanol. Open the incubator and pick up your order. If your order is on a shaker DO NOT TURN OFF THE SHAKER when picking up your order. All other mammalian cell line orders will be in incubator 3. 

Q: Do I need to bring anything when picking up my order? 

A: EHS requires that you bring a secondary container to place any live cells. If you are coming to pick up frozen cells, please bring dry ice. If you forget, we will direct you to the stock room on the ground floor where you can pick up dry ice. 

Q: Do I need a code to get in? 

A: You will need a code to get in before 9am and after 5pm. If you do not have a door code, please email cellculture@berkeley.edu

Q: When can I access items in the freezer?

A: Before 3pm M-F. 

Q: Can I store my cell line with you? 

A: Yes. However, all cell lines must be mycoplasma tested. Please follow the mycoplasma screening protocol on our website. 

Q: What cell lines are available? 

A: Please refer to our list of cell lines on our website under our “Available Cell Lines and Media” section. 

Q: What media do I use for my cells? 

A: Please refer to our list of cell lines on our website under our “Available Cell Lines and Media” section for required media.

Troubleshooting 

Q: Why are my cells dying after a thaw?

A: There are a number of reasons for unsuccessful thaws. 

1. Media: Certain cell lines are very sensitive to the type of media being used. If the media isn’t exact, they could die. Please make sure your cells are being thawed in the proper media and check our xlsx file under Available Cell Lines for media conditions.

2. They’ve been left in DMSO: DMSO is a cryopreservative that can be toxic to cells when left thawed for too long. Our thawing protocol is designed to get rid of DMSO expeditiously since cells are quite sensitive when recently thawed. If not followed correctly, cells can begin to die. Cells are frozen in 10% DMSO so adding our cells to 9 mls of media only dilutes the DMSO to 1% which can still be toxic to cells. Best practice is to pellet the cells and resuspend in fresh media.

3. Contamination: Yeast and bacteria are common contaminants when thawing cells. Note that our protocol is also designed to help mitigate risk of contamination. 

4. Incubator: CO2 and temperature levels are critical for fresh thaws. Please make sure that the CO2 is circulating properly, at 5% and the temperature is at 37 degrees Celsius when placing your cells in the incubator. 

5. If you’ve checked these four things and none of them seem to be the issue, you can reach us directly through Second Lab Messages.

Q: Why aren’t my cells lysing after viral transfection?

A: Although it is rare, it does occasionally happen. As long as your cells are still producing the desired protein, the protein should still be usable.