Good day, my question is fairly easy and I just want to be very careful for my cultured cells. My cells required 90% FBS and 10% DMSO as freezing media. For this, I would need to add the following for each 1mL cryovials?
-900 uL of FBS to each cryovial
-volume of desired cells
-Lastly, 100 uL of DMSO?
I centrifuge my cells, get my pellet, resuspend with 1mL of RPMI 1640, count them to check for viability. Since the RPMI 1640 media has 10% FBS, does this mean the freezing media is actually 800uLs?
Hola Eduardo,
Mi sugerencia es la siguiente:
Cuenta tus células y después las centrifugas (así conoces el numero de celular que tienes y decides la cantidad a congelar en cada vial). Eliminas completamente el medio y le agregas el FSB para que posteriormente le agregues el DMSO. Procedimiento: Por ejemplo si tienes 20 millones de células y vas a congelar 10 millones en cada tubo (2 criovial) entonces agregas 1.8 ml de FSB y resuspendes las células con a pipeta. Después Agregas 900 ul de la suspensión celular en cada criovial y posteriormente a cada uno le agregas los 100 ul de DMSO cierras el tubo mezclas por inmersión. Después haces lo mismo con el siguiente tubo. Si tienes el Mr Frost ahí las guardas y lo llevas a -70 grados C. Tus células se van a congelar y van a mantener una buena viabilidad.
La descongelaron es muy importante ya que un mal procedimiento puede darte viabilidad baja.
Saludos
Your question, which is in the very last two sentences, is not clear.
Regardless, I never have found a cell type that requires 90%FBS to go with the standard 10% DMSO. In my opinion this is an absurd waste of money. A final concentration of 20 to 40% FBS has been sufficient for every cell type that I have frozen.
If you do routinely resuspend your cell pellet in 1 ml of 10% media, then you could add 1 ml of 2x freeze media to it. The 2x freeze media would consist of 20%DMSO and 80% FBS. The final concentration then would be 10% DMSO, 45% FBS, and 45% RPMI.
María Teresa Herrera: Thank you very much for the procedure! Very simple and straight to the point. If for example, I resuspend my cell pellet with FBS and I want to sub-culture 1 million cells into a new flask while freezing the rest of my stock, do I need to centrifuge and wash the new passage cells to take off the excess of FBS?
Ron Hrstka: Thank you for your input. I am going to work with THP-1 X Blue CD14 cells which requires 90% FBS and 10% DMSO for freezing media as stated by the data sheet.
I have found that most data sheets for cell lines are the product of history rather than experimentation. No cell line that I am aware of needs to be cultured in 90% FBS and I have yet to find one that needs to be frozen in it; yet that 90:10 ratio remains the standard without any good reason.
Hello! I usually freeze my cells in a total volume of 1ml for each criotube. My medium is composed of 50% complete growth medium, 40% FBS and 10% of DMSO. I add the DMSO in complete medium, resuspend my pellet in 40%of FBS (400ul for each criotube) and then add the rest 600ul (composed of medium+DMSO) toreach the final volume of 1ml.
Dear Eduardo,
Respect your question.
First, you should count your total cells then you separe the volumen of the cells suspention (corresponding to 1 million) that you want to work and the rest of cells you centrifuga and continue with the freezing protocolo.
My experience is with human PBMC and THP1 and the resuts are good.
Bye
Dear Ron, it is not a waste of money when your cells are very important, there are cells like THP1 needs a culture medium with FBS f (not freezing) and it is a requerid by the cells.
Geranios
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