3 replies to this topic

[Chelo]

Dear forum members,

I am new in the field of immunology. In our lab, we make monoclonal antibodies by hybridoma production. I have a question that could not be answered by my colleagues so that is why I would like to try it in this forum.
For the step of lymphocytes and myeloma cells fusion there are different protocols available. In all of them there is an excess of lymphocytes mixed with myeloma cells (depending on the protocol, the proportions go from 2:1 up to 10:1). My specific question is why the lymphocytes are in excess. In other words, if the myeloma cells are limiting, there will be some/many potential valuable B-cells which won´t find a partner to fuse with. I think we lose the chance to get more hybridomas! Yet, as I said, I didn´t find any protocol where more myeloma cells are used in excess.
Does anyone know the logics behind this?
Thank you very much in anticipation!

[casandra]

Chelo, on 08 April 2011 - 02:43 AM, said:

Dear forum members,

I am new in the field of immunology. In our lab, we make monoclonal antibodies by hybridoma production. I have a question that could not be answered by my colleagues so that is why I would like to try it in this forum.
For the step of lymphocytes and myeloma cells fusion there are different protocols available. In all of them there is an excess of lymphocytes mixed with myeloma cells (depending on the protocol, the proportions go from 2:1 up to 10:1). My specific question is why the lymphocytes are in excess. In other words, if the myeloma cells are limiting, there will be some/many potential valuable B-cells which won´t find a partner to fuse with. I think we lose the chance to get more hybridomas! Yet, as I said, I didn´t find any protocol where more myeloma cells are used in excess.
Does anyone know the logics behind this?
Thank you very much in anticipation!

well, don't get so excited Chelo, ok, but I think that you normally use an excess of the mouse lynphocytes to compensate for their short life span in culture (they survive only for a few days and start dying off) while the myeloma cells would still continue dividing before you add the HAT selective medium....

Edited by casandra, 08 April 2011 - 07:28 AM.

[Chelo]

casandra, on 08 April 2011 - 07:24 AM, said:

Chelo, on 08 April 2011 - 02:43 AM, said:

Dear forum members,

I am new in the field of immunology. In our lab, we make monoclonal antibodies by hybridoma production. I have a question that could not be answered by my colleagues so that is why I would like to try it in this forum.
For the step of lymphocytes and myeloma cells fusion there are different protocols available. In all of them there is an excess of lymphocytes mixed with myeloma cells (depending on the protocol, the proportions go from 2:1 up to 10:1). My specific question is why the lymphocytes are in excess. In other words, if the myeloma cells are limiting, there will be some/many potential valuable B-cells which won´t find a partner to fuse with. I think we lose the chance to get more hybridomas! Yet, as I said, I didn´t find any protocol where more myeloma cells are used in excess.
Does anyone know the logics behind this?
Thank you very much in anticipation!

well, don't get so excited Chelo, ok, but I think that you normally use an excess of the mouse lynphocytes to compensate for their short life span in culture (they survive only for a few days and start dying off) while the myeloma cells would still continue dividing before you add the HAT selective medium....

Dear Casandra, the lymphocytes are normally collected the very same day of the fusion. So their short life span shouldn´t be an issue. There must be some other reasons for the different proportion of cells.

[casandra]

Chelo, on 08 April 2011 - 12:21 PM, said:

casandra, on 08 April 2011 - 07:24 AM, said:

Chelo, on 08 April 2011 - 02:43 AM, said:

Dear forum members,

I am new in the field of immunology. In our lab, we make monoclonal antibodies by hybridoma production. I have a question that could not be answered by my colleagues so that is why I would like to try it in this forum.
For the step of lymphocytes and myeloma cells fusion there are different protocols available. In all of them there is an excess of lymphocytes mixed with myeloma cells (depending on the protocol, the proportions go from 2:1 up to 10:1). My specific question is why the lymphocytes are in excess. In other words, if the myeloma cells are limiting, there will be some/many potential valuable B-cells which won´t find a partner to fuse with. I think we lose the chance to get more hybridomas! Yet, as I said, I didn´t find any protocol where more myeloma cells are used in excess.
Does anyone know the logics behind this?
Thank you very much in anticipation!

well, don't get so excited Chelo, ok, but I think that you normally use an excess of the mouse lynphocytes to compensate for their short life span in culture (they survive only for a few days and start dying off) while the myeloma cells would still continue dividing before you add the HAT selective medium....

Dear Casandra, the lymphocytes are normally collected the very same day of the fusion. So their short life span shouldn´t be an issue. There must be some other reasons for the different proportion of cells.

Dear Chelo, precisely....you collect them on the very same day of the fusion bec many won't be as viable if you collect them before and keep them in medium indefinitely...and then you'd normally use PEG(?) for fusion which would also kill off a certain percentage of your spleen cells...and considering that myeloma cells are adapted to cell culture and had been manipulated here and there (well- maintained and happily waiting to be fused), they're more robust and therefore have a better chance of surviving the procedure and proliferating afterwards.....so if you want to increase the proportion of spleen cells fusing successfully and eventually producing hybrids, you've have to add them in excess...