How To Work With ATCC Cells?

Congratulations on receiving your ATCC cells!

This instruction manual is NOT intended to substitute reading the cells’ product sheet and certificate of analysis!!!

In order to ensure success in culturing your cells there are several issues you should be aware of:

1. ATCC warranty is for 30 days from receipt of cells, so hurry up and thaw the cells as soon as possible.
2. Once cells are received either thaw on the same day or place in a liquid nitrogen tank (DO NOT STORE CELLS IN -80°C)
3. In the first thawing of ATCC cells you must use media purchased from ATCC.
4. After freezing an initial cell bank, you may opt to use similar formulation media from alternative media suppliers. We can help you find the most suitable more economical alternative from BioWest options.
5. Unless mentioned otherwise, you must use regular, non heat-inactivated, serum.
6. You may use antibiotics- please use only penicillin/streptomycin at maximal final concentrations of 100 U/ml penicillin and 100µg/ml streptomycin

Instructions for thawing the cells you received:

1. Go to ATCC website (atcc.org)  and open the page of the cells you ordered
2. Scroll down to Detailed product information

3. Press “Expand all”
4. Go to Handling information, See growth conditions recommended by ATCC

5. Before thawing cells make sure you have every product needed to:
   1. prepare growth medium
   2. dissociate the cells as specified in ‘subculturing procedure’
   3. cryopreserve the cells as specified in ‘ reagents for cryopreservation’

6. Open the Certificate of analysis (COA) by entering the item lot# (מס’ מנה/סדרה) as appears in the delivery certificate (תעודת משלוח) you received

7. Within the COA you can find important information such as:
   1. Total number of cells you received
   2. Expected post freeze viability
   3. Recommended recovery procedure (flask size, seeding density and expected time to confluency)

8. Calculate the amount of flasks needed to seed the whole vial based on number of cells/vial, expected post-freeze viability and recommended seeding density.
9. Prepare flasks for cell seeding:
   – In a 25cm² flask put 8-10 ml growth media
   – In a 75 cm² flask put 15-20 ml growth media
   Place flasks with the cap half open within the incubator to allow the medium to reach adequate temperature and pH.
10. If centrifugation to eliminate DMSO is instructed then:
    1. Prepare a 15ml tube with 9 ml of media, place in a 37°C water bath to warm.
    2. Thaw the cells- take cell vial out of the liquid nitrogen tank and place immediately in a 37°C water bath with gentle agitation until a few ice crystals remain (it should take ~2 minutes).
    3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
    4. Transfer the vial contents to a centrifuge tube prepared in step ‘a’ and spin at approximately 125 x g for 5 to 7 minutes (it is extremely important to spin at the recommended speed or slightly higher- not more than 150-200 x g). Discard supernatant. Resuspend the cell pellet with 1 ml of the recommended complete medium and dispense into the prepared culture flask/s. 
11. If centrifugation to eliminate DMSO is not recommended simply place the cells (from vial thawed as described in 10 b-c) into the prepared culture flask/s. 

Instructions for subculturing the cells you received:

Read the instructions for subcuturing under ‘subculturing procedure’ in the ‘handling information’ tab

For adherent cells:
*It is recommended to split cells when they reach 80-90% confluence
*If many floating cells are present, it is recommended to examine floating cell viability using trypan blue, if most of the cells are dead, you can discard them, otherwise transfer them back to the growing culture (not in a separate flask). Do this only for the first two subcultures.

1. Remove culture medium
2. Wash cells with PBS w/o Ca⁺⁺/Mg⁺⁺
3. Add appropriate volumes (1-2ml for 25 cm² flask; 2-4ml for 75cm² flask) of appropriate trypsin/EDTA solution (as mentioned in product sheet- it is imperative to use a trypsin solution with similar composition to the one recommended on the website).
4. If centrifugation is recommended Transfer the cell suspension to the centrifuge tube, and centrifuge at approximately 125 x g for 5 to 10 minutes. Discard the supernatant. Sometimes the use of a trypsin inhibitor or a trypsin neutralization solution is also needed.
5. Split according to the subcultivation ratio/ seeding densities recommended.
6. Make sure to count your cells and determine % viability every time you split the cells, this will ensure you do not over/under dilute them when splitting

*** Even if cells are not ready for splitting, make sure to renew their medium every 2-3 days

For suspension cells:
* it is strongly recommended to do daily counts of the cells, maintaining culture densities as recommended under ‘subculturing procedure’ in the ‘handling information’ tab

• When splitting suspension cells it is NOT recommended to centrifuge the cells. It is preferred to split the cells into the desired number of flasks and supplement with fresh media to reach optimal growth volume.
• *** Even if cells are not ready for splitting, make sure to renew their medium every 2-3 days- do this also without centrifugation- let the flask stand vertically for 15-30 minutes, so the cells will sink to the bottom and remove 50-75% of the media (w/o disturbing the cells on the bottom most part of the flask) and replace with fresh media.

Instructions for cryopreservation of the cells you received:
For adherent cells:

1. Remove culture medium
2. Wash cells with PBS w/o Ca⁺⁺/Mg⁺⁺
3. Add appropriate volumes (1-2ml for 25 cm² flask; 2-4ml for 75cm² flask) of appropriate trypsin/EDTA solution (as mentioned in product sheet- it is imperative to use a trypsin solution with similar composition to those recommended on the website).

For adherent cells & suspension cells:

1. Count the cells and determine viability
2. Transfer the cell suspension to the centrifuge tube and centrifuge at approximately 125 x g for 5 to 10 minutes. Discard the supernatant.
3. Resuspend the cells in appropriate volume of cell cryopreservation solution. Freeze cells at similar concentrations to those received from ATCC or as needed for post-thaw experiments. It is usually not recommended to freeze less than 0.5M cells/ml or more than 10M cells/ml.
4. Unless otherwise mentioned under ‘reagents for cryopreservation’ in the ‘handling information’ tab, the recommended cryopreservation media is Complete growth medium supplemented with 5% (v/v) DMSO.
5. Following cell suspension in cryopreservation medium, place the vial in an appropriate pre-cooled (4°C) cryopreservation container (i.e. CoolCell® LX Alcohol-Free Cryopreservation Container) in the -80°C freezer for slow temperature reduction, for at least 24 hours (maximum 7 days).
6. For long-term storage Transfer the cells to a liquid nitrogen tank. Do not store cells for more than a few days in the -80°C freezer.

GOOD LUCK 😊

Call me for any issue that may arise:
Dr. Shraga-Heled Niva 052-4212211