2 replies to this topic

[vivianchan]

Hi all,
   I am having problem with monoclonal antibody. The fusion partner I am using is SP2/0-ag14 (ATCC:1581) and I am growing it in DMEM+10%FBS+ Penicillin:streptomycin. The SP2/0 seems normal (round suspension cells) until I perform fusion! After I performed fusion and added HAT medium, the morphology of the cells change to fibroblast-like (please see attached pictures!) I am sure this strange cells are Sp2/0 as I have encounter this before in another lab and  fusions using NS0 fusion cell lines do not produce this strange cells! Anyone know what's wrong with my SP2/0????? This strange Sp2/0 is surrounding my hybridoma cells in EVERY wells and I am suspecting it may be the reason why I have only little hybridoma cells growing! (none of them is positive!!!!!)
    Please help me as I am really struggling to get a monoclonal antibody! (I cant change the fusion partner cell line as I dont have money to buy a new one ><) Is this strange morphology normal to Sp2/0???? (I have seen it before in another lab) or is it because something wrong with my medium? but my sp2/0 grow well in dmem usually, just except after fusion that it go strange! I suspect the HAT medium as well but normal unfused SP2/0 just die so I dont know where this strange cells come from?

Any advise are greatly appreciated! Thanks a lot in advance!

Best Regards,
Vivian

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Edited by vivianchan, 29 August 2010 - 11:31 PM.

[Roo]

Hi Vivian,

Yes, we do have sp2/0 cells, but we never got around to using them.  We usually use NS-1 cells, but continually had problems so we bought some sp2/0 cells.  However, we stopped our antibody work at that time so we never ended up using  them.  Needless to say, this means I can't tell you whether their fibroblast like morphology is normal, but that doesn't sound right to me.  I would recommend that you contact the ATCC since they sell this cell line.  Tell them you are using sp2/0 cells and what is happening.  They should be able to tell you if this is normal or not.  The ATCC contact info is below:

American Type Culture Collection (ATCC)
P.O. Box 1549
Manassas, VA 20108
USA

Contacting Technical Support:
You may call ATCC Technical Support from 9:30am-4:30pm ET Mon.-Fri. With our new customer support system, your ATCC account number or complete organization name and address may be necessary for technical inquiries.

http://www.atcc.org/...83/Default.aspx (Technical support digital submission page)

Sp2/0-Ag14                                                              ATCC Catalog # CRL-1581  
Mus musculus (B cell); Mus musculus (myeloma) (mouse (B cell); mouse (myeloma))  


A couple of questions though:

1.  Did you pass your sp2/0 through 8-azaguanine before using them for fusion?  
This doesn't have to be done every time, but we periodically pass our NS-1 (or whatever myeloma line we're using) through 8-azaguanine to ensure that they are HGPRT negative/8-azaguanine resistant.  Otherwise, they will not be sensitive to the aminopterin in HAT.  My guess is that this isn't the problem because then you would be overrun with myeloma cells regardless of whether they had fused or not.

2. Are you 100% you didn't get fibroblasts (or macrophages) in your primary culture when harvesting/culturing the spleen cells?  
If you get too much connective tissue from the spleen, you will end up with fibroblasts in your culture. We have encountered a few fibroblasts in our cultures on occasion, but this should be minimal.  We usually just omit these wells since it is such a small number of the 10 - 20  96-well plates we use when making hybridomas.

Best wishes are solving your problem!

[vivianchan]

Dear Roo,
    Thank you very much for your reply ^^ I have emailed ATCC and hopefully they can tell me if it is something wrong with my culturing condition or problem with cells!

For your questions  
    I did use 8-azaguanine for passing the Sp2/0 before fusion and I have tested that all Sp2/0 die in HAT before I go to fusion.
    In my previous lab I have encounter the same problem and the fibroblast-like morphology only happens in fusion using Sp2/0 but not in fusion with NS0.......(such a pity that I could not get the NS0 from my previous lab ^______^" ) So i guess it is not caused by too much connective tissue from the spleen. But next time I will make sure there's no connective tissue and see if it can solve the problem

Thank you very much for you help! It's very kind of you.

Best Wishes,
Vivian

  

Roo, on 30 August 2010 - 07:42 AM, said:

Hi Vivian,

Yes, we do have sp2/0 cells, but we never got around to using them.  We usually use NS-1 cells, but continually had problems so we bought some sp2/0 cells.  However, we stopped our antibody work at that time so we never ended up using  them.  Needless to say, this means I can't tell you whether their fibroblast like morphology is normal, but that doesn't sound right to me.  I would recommend that you contact the ATCC since they sell this cell line.  Tell them you are using sp2/0 cells and what is happening.  They should be able to tell you if this is normal or not.  The ATCC contact info is below:

American Type Culture Collection (ATCC)
P.O. Box 1549
Manassas, VA 20108
USA

Contacting Technical Support:
You may call ATCC Technical Support from 9:30am-4:30pm ET Mon.-Fri. With our new customer support system, your ATCC account number or complete organization name and address may be necessary for technical inquiries.

http://www.atcc.org/...83/Default.aspx (Technical support digital submission page)

Sp2/0-Ag14                                                              ATCC Catalog # CRL-1581  
Mus musculus (B cell); Mus musculus (myeloma) (mouse (B cell); mouse (myeloma))  


A couple of questions though:

1.  Did you pass your sp2/0 through 8-azaguanine before using them for fusion?  
This doesn't have to be done every time, but we periodically pass our NS-1 (or whatever myeloma line we're using) through 8-azaguanine to ensure that they are HGPRT negative/8-azaguanine resistant.  Otherwise, they will not be sensitive to the aminopterin in HAT.  My guess is that this isn't the problem because then you would be overrun with myeloma cells regardless of whether they had fused or not.

2. Are you 100% you didn't get fibroblasts (or macrophages) in your primary culture when harvesting/culturing the spleen cells?  
If you get too much connective tissue from the spleen, you will end up with fibroblasts in your culture. We have encountered a few fibroblasts in our cultures on occasion, but this should be minimal.  We usually just omit these wells since it is such a small number of the 10 - 20  96-well plates we use when making hybridomas.

Best wishes are solving your problem!