Depends on the cell line, the freezing procedure are specifically mentioned in spec-sheet provided by ATCC after purchase. Normally in secondary cultures, we freeze in freezing media containing 5% DMSO in working media (10%FBS+plain media+supplements (if any)).
the earlier the passage the better. if you thaw a cell type with a low passage number, and that cell stock is less than 2, then you make more aliquots of that low passage number cell stock
Passage number alone isn't the only indicator for freezing cells in culture. Here's what matters most: • Cell health and viability: Cells should be actively growing, with high viability, usually >90%. (You can look for minimal cell death and signs of stress.) • Exponential growth phase: Ideally, freeze cells during the exponential growth phase when they are rapidly dividing. (By doing this you ensure a good yield for future experiments.) • Low passage number: While not a strict rule, freezing cells at a lower passage number (generally between 2-10) is preferred. (It minimizes the risk of mutations or accumulated cellular changes that can occur with repeated passaging.) I hope this helps you!
Hi Harry, Thank you for your question. No, cells should be kept in an isopropanol chamber at -80°C for overnight prior to liquid nitrogen storage. For more information about freezing cells, please check the following link. Thank you! www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/freezing-cells.html.
Thank you for your question Qin. The amount of freezing media will be dependent on two factors: the culture vessels (example: Cryo vials) that is being used and the density of the cells. The volume often ranges from 0.5 mL to 4 mL. For additional questions, please contact our Technical Support team at thermofisher.com/askaquestion and we’d be happy to help.
After trypsinization (detaching cells), they spin down the culture in a centrifuge to pellet the cells at the bottom. The supernatant (liquid on top) gets discarded because it contains: • Trypsin: This enzyme detaches cells but can be harmful if left on during freezing. • Dead cells & debris: These won't survive cryopreservation and can harm healthy cells. They want healthy, live cells for freezing, so they keep the cell pellet and wash away the rest!
Thank you for your question Nimrah. Can you please reach out to our technical support team so they can reply to your question? They can be reached at thermofisher.com/askaquestion. Thank you!
I am assuming you the +/+ notation is for the presence of calcium and magnesium ions. For washing purposes calcium and magnesium (+/+) are generally added to the PBS.
Thank you for your question. If the freezing media is made in house (ie in your lab), we do recommend filtering the media. However, if you purchase an already made freezing media from us, there is no need to filter. For additional information, please contact our technical support team at thermofisher.com/askaquestion. Thank you!
Addition of cryoprotectant in freezing cell (like DMSO) is missing.
Umihealth soutwoods
Freezing medium contains DMSO
Isn't it usually 90% fbs 10% dmso?
Depends on the cell line, the freezing procedure are specifically mentioned in spec-sheet provided by ATCC after purchase.
Normally in secondary cultures, we freeze in freezing media containing 5% DMSO in working media (10%FBS+plain media+supplements (if any)).
You are rigth!
How do you know when your cells are ready to be frozen? Is it at a specific passage number?
the earlier the passage the better. if you thaw a cell type with a low passage number, and that cell stock is less than 2, then you make more aliquots of that low passage number cell stock
Passage number alone isn't the only indicator for freezing cells in culture. Here's what matters most:
• Cell health and viability: Cells should be actively growing, with high viability, usually >90%. (You can look for minimal cell death and signs of stress.)
• Exponential growth phase: Ideally, freeze cells during the exponential growth phase when they are rapidly dividing. (By doing this you ensure a good yield for future experiments.)
• Low passage number: While not a strict rule, freezing cells at a lower passage number (generally between 2-10) is preferred. (It minimizes the risk of mutations or accumulated cellular changes that can occur with repeated passaging.)
I hope this helps you!
Thank you for this video series. It helps so much :)
Hello katarinamajerikbehinska, Glad it was helpful!
*Can we preserve cells in -70 C for long term storage?*
Hi Harry,
Thank you for your question. No, cells should be kept in an isopropanol chamber at -80°C for overnight prior to liquid nitrogen storage. For more information about freezing cells, please check the following link.
Thank you!
www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/freezing-cells.html.
You could use a Freeze dryer
How do you determine the appropriate volume of freezing media?
Thank you for your question Qin. The amount of freezing media will be dependent on two factors: the culture vessels (example: Cryo vials) that is being used and the density of the cells.
The volume often ranges from 0.5 mL to 4 mL. For additional questions, please contact our Technical Support team at thermofisher.com/askaquestion and we’d be happy to help.
Das sieht sehr interessant aus!
येस
bonjourno
Why was the culture centrifuged after trypsinization and the supernatant discarded?
you need to change their medium before freezing. If you use DMSO the solution won't form crystals (like water does) and break the cells
After trypsinization (detaching cells), they spin down the culture in a centrifuge to pellet the cells at the bottom. The supernatant (liquid on top) gets discarded because it contains:
• Trypsin: This enzyme detaches cells but can be harmful if left on during freezing.
• Dead cells & debris: These won't survive cryopreservation and can harm healthy cells.
They want healthy, live cells for freezing, so they keep the cell pellet and wash away the rest!
Did you use PBS -/- or +/+ for washing cells? Thank you
Thank you for your question Nimrah.
Can you please reach out to our technical support team so they can reply to your question? They can be reached at thermofisher.com/askaquestion.
Thank you!
I am assuming you the +/+ notation is for the presence of calcium and magnesium ions. For washing purposes calcium and magnesium (+/+) are generally added to the PBS.
What is the freezing media?
Thank you for your question. Please contact our technical support team at thermofisher.com/askaquestion for additional information. Thank you!
L929?
Yes, our cell freezing medium can be used with L929 cells.
is this necessary to filter the freezing medium?
Thank you for your question. If the freezing media is made in house (ie in your lab), we do recommend filtering the media. However, if you purchase an already made freezing media from us, there is no need to filter. For additional information, please contact our technical support team at thermofisher.com/askaquestion. Thank you!
what kind of youtube rabbit hole have a stumbled upon bruh
welcome to the nerds world