You have probably thought through all of the below, but for the record, here is a list of things to think about: **8 yrs is a long time, but PEG should be fine if stored properly. Do you buy it sterile or do you autoclave before use in fusion medium? If the latter, had it been allowed to cool to RT before use?

**How did you homogenize the spleen? If you used a hand-held device, could the process have been too aggressive? Did you achieve a single cell suspension once the spleen was homogenized?

**Are you sure your fusion partner was fine - no mycoplasma? in log phase? high viability? added to spleen cells @ an optimal ratio?

**Did you use an electrofusion device or just use centrifugation? Any chance that the settings were incorrect whichever device you used?

**When you plated the putative hybridomas, are you sure the selection agent was added correctly? What did you use to plate with - multi-channel pipettor, automated system? The bore size of whatever you use can sometimes make a difference, or the force behind what expels the cells from the tip or pin cam make a difference?

**Did nothing at all grow out? How long has it been? Sometimes it can take up to 4 or 5 weeks for hybridomas to grow out. During that time, the medium should only be replenished b/c of evaporation, but otherwise should not be added to per se nor changed out for 'fresh' medium.

**Did you plate the 'entire' fusion, in how many plates, and what size well in the plate? If you overload or under-seed the wells, it can be an issue. Also, if gp hybridomas are difficult to generate and you elected not to plate the entire fusion vol., you could have missed that rare event(s).

**If something did grow out, but your screen turned up no positives, those reagents and instruments have to be considered in the troubleshooting process.

**Finally, is your CO2 incubator at the right temp, delivering the correct amount of CO2, and properly humidified?

As far as I know, no one has been able to raise guinea pig monoclonals

We worked on the hybridomas fusion from mouse many times and despite the high percentage of successfull experiments there was a little percentage of fusions where no hybridomas was obtained. Unfortunatly is the norm, at list as regard the mouse model.

You have probably thought through all of the below, but for the record, here is a list of things to think about: **8 yrs is a long time, but PEG should be fine if stored properly. Do you buy it sterile or do you autoclave before use in fusion medium? If the latter, had it been allowed to cool to RT before use?

**How did you homogenize the spleen? If you used a hand-held device, could the process have been too aggressive? Did you achieve a single cell suspension once the spleen was homogenized?

**Are you sure your fusion partner was fine - no mycoplasma? in log phase? high viability? added to spleen cells @ an optimal ratio?

**Did you use an electrofusion device or just use centrifugation? Any chance that the settings were incorrect whichever device you used?

**When you plated the putative hybridomas, are you sure the selection agent was added correctly? What did you use to plate with - multi-channel pipettor, automated system? The bore size of whatever you use can sometimes make a difference, or the force behind what expels the cells from the tip or pin cam make a difference?

**Did nothing at all grow out? How long has it been? Sometimes it can take up to 4 or 5 weeks for hybridomas to grow out. During that time, the medium should only be replenished b/c of evaporation, but otherwise should not be added to per se nor changed out for 'fresh' medium.

**Did you plate the 'entire' fusion, in how many plates, and what size well in the plate? If you overload or under-seed the wells, it can be an issue. Also, if gp hybridomas are difficult to generate and you elected not to plate the entire fusion vol., you could have missed that rare event(s).

**If something did grow out, but your screen turned up no positives, those reagents and instruments have to be considered in the troubleshooting process.

**Finally, is your CO2 incubator at the right temp, delivering the correct amount of CO2, and properly humidified?

I have never gotten guinea pig fusions to work wioth high efficiency. The SP 2/0 myeloma cells are a mouse cell line and do not work well as a fusion partner for guinea pig B cells. I do not know of any guinea pig myeloma or plasmacytoma cells that are available, but these are what you need to look for. Otherwise, using some normal guinea pig sera along withthe fetal bovine serum. Be sure to use only heat inactivated sera as guinea pigs have a very high level of complement in their sera.

I don`t have any idea aboutguinea "pig" fusions but, I hope to be patient,the hybridoma generation is always difficult and need attention plus chance.

Hope you succeed to get the stable clones.