hi

It is natural because it is unstable clones.

1- screening your all clones with more precision.

2- before run the fusion, check serum specific igG titter ( OD > 2)

3- you can used semi-solid medium for Hybridoma cell cloning .

4- check your cells for mycoplasma infection .

good luck

https://www.stemcell.com/clonacell-hy-96-well-plate-semi-solid-cloning-lp.html

I think as Reza Hadavi in his 3rd suggestion of using semi-solid medium for hybridoma cell cloning. 

It would be useful too to add feeder cells as fibroblast to enhace the proliferation of your clones.

Your cultures must be controled microscopically each day to eliminate contamination, cell death or the nitrogen content of your culture cell incubator.

The electrical shutdown in your lab must be also prevented with a backup system.

Thank you SIr for your nice suggestions... I will try semi sold media for for proliferation and get back to you .........Thanks alot

The suggestions above are good. I can add a couple of things to try or test.

1. Post HAT selection, you do need to wean the cells by using HT medium [no aminopterin] for several days, then 1/2 HT medium for several days, then 1/4 HT medium for several days. After that you can culture the cells in regular growth medium. This is to allow the cells to 'de-tox' after aminopterin selection.

2. In limiting dilution, there are several factors that can lead to clonal loss/death. One problem is loss of the amino acids serine and glycine from the cells. This can be averted by supplementing your medium with 100 mM sodium pyruvate.

3. Hyrbidoma cloning efficiency can be significantly improved by adding a hybridoma growth supplement. It is basically conditioned medium that contains IL-6 and other factors. The best stuff is produced by Origen. 

4. Pertioneal macrophages [isolated by lavaging the abdomen of a BALB/c mouse with ~ 50 mL DMEM medium, then carefully collecting as much as you can without puncturing the bowel. plate out overnight ~ 0.1 ml/well of 24 well plate] can be used as a feeder layer under the methylcellulose semi-solid medium.

5. Check your fetal calf serum for mycoplasma contamination. Mycoplasma can make hybridomas lose Ig production.

5 ml, not 50.

I agree with Gary. We always use sodium pyruvate in our cell culture medium, and supplement the cloning medium with IL-6.  On a typical 96-well plate, how many of your wells have single clones? You may want to prepare more than one plate to increase your chances. Also, always remember to screen the parent clone at the same time, it's not unusual for a parent clone to stop secreting. 

Thank You every one for your nice suggestions.

Dear Gary Sir and Daad Mam, I have already reduced HT medium (1 week 1/2 HT; 1 week 1/4 HT and etc..) from the hybridoma clone, as you have suggested. Presently, hybridoma clones are growing in HT free medium (RPMI,1640 with 10% FBS). But,  I am not sure about the concentration of sodium pyruvate  in the RPMI medium, because i am using commercial RPMI (Hyclone,1640, cat no-SH30027.01). 

Additionally, we have plated feeder cells one day before fusion.  1X hybridoma growth supplements was used during hybridoma clone development ( 1st plating after fusion with PEG) and plated out newly fused cells to the 24 well TC plates (4 plates for each mice).

Now, i have little confusions on some points..

1. How many sub-cloning is necessary for finding of true hybridoma clone.

2. How many days clones must be grow in the HT medium, Post HAT selection.

3. Can be use ciprofloxacin for removing of mycoplasma contamination..if present in the FBS..Al through, company has claimed free of mycoplasma form FBS.

Once again Thank you every one...

You all seems to be forgetting something very basic: when we clone hybridomas, we will get A LOT of negative clones, simply because the cell that was dropped on those wells was not the hybridoma cell that was producing the antibody specific against the immunogen. Yes, thats right, pure bad luck! Therefore, it is totally normal to get negative results at the screening at cloning (or even sub-cloning, if the first cloning was not successful in delivering a true single-cell clone). Here in my lab I instruct my students to perform 4x 96well plates for each positive hybridoma to be cloned. My rate of success in isolating real positive clones is 15%.

This is true, but you should see SOME positives on your LD plates. Hybridomas are inherently unstable initially because of the hyperploid nature of the initial fusion [normal mouse cells have40 chromosomes, most myelomas have over 60] so chromosomal loss is common and random.

Anil - To get a stable clone, I typically do 2 rounds of LD. The first round typically has 10-15%+ wells, but the second round should be >85%. If not, I do a third round.

I used to study short heavy chain variants in myeloma, and those are quite unstable. I had to subclone one line 5 times. 

RPMI medium does not normally contain pyruvate, It is added as a supplement.

In my experience, cipro will suppress mycoplasma, but it will nor cure it. The gold standard is to passage the hybridoma through a pristine-rimed mouse, and collect the ascites aseptically, Spin down the cells and plate them out in normal medium.

As to the timing of HT-->1/2HT-->1/4HT-->no HT, my recollection is a week for each step.

Thank you Dr Gary and Dr Andrei for your nice suggestions. I was successfully sub cultured and  isolated stable hybridomas. Now, i am struggling with leveling of monoclonal antibody  with HRP. I have tried so  many elution buffers like glycine, sodium acetate, sodium citrate. but could not achieve HRP leveled Antibody.

Thank you every one for helping me ..