Problem with Monoclonal antibody production - why cells die after adding HAT?????? (Jul/30/2007 )

Hi there,
I have been trying to produce hybridoma for one whole year without success. No cell survival was observed 10 days after addition of HAT medium! the cell survive if I grow them in HT medium but not when I add HAT!!! Does it mean that the fusion was not successful? Is there any tricks that can help me to solve the problem?

I have been using both NS1 and SP2/0 cell line, neither of them produce hybridoma that survive in HAT.

the peg i am using is (sigma p7306 ) Hybri-Max™, 50 % (w/v) in 10% aqueous DMSO (v/v), average mol wt 1,450, sterile-filtered, hybridoma tested

Below is my protocol, can anyone help me and see if there's anything wrong so I can improve? Thank you very very much! I am stucked and I really need help! Thanks in advance! ^^

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The mouse is immunized six times

Booster 3 days before fusion : just peptide (40ug)

1 days before fusion
Split SP2/0 1 in 3 in 20% RPMI medium. Centrifuge at 60g for 2 minutes (3x T150 flask)


Fusion Day:

Dissection
 Kill the mice by cervical dislocation, Soak the fur in ethanol
 Remove spleen in 5-10ml plain RPMI
 Wash in plain RPMI in a culture dish.
 Place mesh over a culture dish and half fill with plain RPMI. Transfer the spleen and mesh it gently. (with a plunger)
 Transfer cells to falcon tube.
 Pellet cells (2000rpm for 5 minutes) ,
 Wash once in ice cold plain RPMI and resuspend in 5ml plain RPMI
 warm cells (take 10ul to count and warm the rest)

Prepare SP 2/0 cells
 Centrifuge down cells from 3X150 flask => 1000g for 5 minutes
 Wash once in plain RPMI
 Resuspend cells in 5ml plain RPMI. warm cells (take 10ul to count and warm the rest)
 Count both spleen cells and SP 2/0 cells.

Fusion
 Mix cells (5 spleen cells to 1 SP2/0 cells) – pool all spleen cells then add SP2/0, can vortex 
Centrifuge at 1000rpm for 5 minutes to pack cells
 Wash cell mixture twice with warm 20ml plain RPMI.
 Remove ALL supernatant (even with some lost of cells)
 Break the pellet by tapping
 Add 1ml warm 50% PEG over 1 minute. (gently stir the cells and break large crumps)
 Stir for 1 minute,
 Gently add 3ml warm plain RPMI over 3 minute period. Add the media as gentle drops.
 Add 10ml plain RPMI over the next 2 minutes.
 Add 10ml plain RPMI over 1 minute.
 Centrifuge cells at 800 rpm (50g) for 5minutes.
 Remove supernatant
 Put cells in 50ml 20% RPMI (with 1 x HT for best result) .
 Dispense cells into 96-well plates (100ul each well) and incubate at 37oC
 The next day, add 100ul 1x HAT DMEM into each well (making it 0.5 x HAT and 0.5x HT)
 Return cells to 37 oC.
 Change medium after 3-4 days
 Hybridomas can be tested by ELISA when they cover approximately one third of the well. 
Assay after 7 and 10 days.
 Change to HT DMEM after 14 days.

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my cells normally die within 10 days! ><
Please help me! Thanks a lot!!!!

Hi leelaram,
Thank you very much for you reply ^^ actually, I use RPMI as medium and I dont know whether it will make any difference if I change to DMEM? but I guess I can try to use IMDM next time just to see if it gives me more luck also, I did do the mixing step while adding PEG and I keep mixing for another one minute just to make sure the cells and PEG are well mixed....

I suspected that the PEG I am using is not the suitable one because I dont know if its a good idea to include DMSO in the PEG? but as you said you are using the same PEG as me so I guess its not the point ^^"

Thank you very much for your help ^^

u07hlc

Hi leelaram,
Thank you very much for you reply ^^ actually, I use RPMI as medium and I dont know whether it will make any difference if I change to DMEM? but I guess I can try to use IMDM next time just to see if it gives me more luck also, I did do the mixing step while adding PEG and I keep mixing for another one minute just to make sure the cells and PEG are well mixed....

I suspected that the PEG I am using is not the suitable one because I dont know if its a good idea to include DMSO in the PEG? but as you said you are using the same PEG as me so I guess its not the point ^^"

Thank you very much for your help ^^

u07hlc

hello,
after going through your protocol, i have couple of hints to let you know which may help you.

1-donot worry too much about DMSO (final conc 0.1%) which is not a cytotoxic concentration.

2- have an aliquote of unfused cells with HAT from the begining. these cells must survive if they die then some thing to do with DMSO or PEG or may be some other contaminants (if any exist) in the product.

3-if they survive then you should think about higher levles of this antibody production may be involving in some process at cellular levle?
for example it may bind to any cell surface proteins after its release in to the medium?
to control this possibility partially u can change medium almost every day.

4- despite all above mentioned possibilities i m more comfortable with p3x63 cells rahter than sp2O as i had some other problems.


gud luk
sravan