I would like to know more about what you mean by 'antibody custumization' before i try to make a suggestion regarding the fixing technique.

Dhouha Daassi Hello

Depending on what you want to detect in these cells the fixation can vary. As for shipping, its not exactly the fixation but the storage that makes the main difference. We have used organic solvent fixation and 4% PFA-PBS fixation and both work well after storage.

For short delivery time- we add 0.1% Azide-PBS to fixed cells and seal the plate. The plates or slides are then packed into a box with ice-coolers and shipped at 4 degrees C. Storage is also at 4C at all times. This way they survive for up to a month.

For long deliveries and long storage ( up to 6 months)- Fixed cells are mounted on semi-solid mounting media (like Fluoromount G), then dried in the dark, sealed in a bag with desiccating agents. Samples are shipped and stored at 4 degrees C at all times. When reviving these plates, we add PBS to the wells to solubilize the mounting agent, then wash it off and use these cells for staining.

Hope this helps

Best

Aparajita

I agree with with Aparajita Lahree.

You can use 4% paraformaldehyde, 3.7% formaldehyde or chilled methanol (-20) as per your assay required.

Ashutosh Acharya antibody custumization means using the cells to immunize an animal and generate antibodies for the specific cell samples we have

Aparajita Lahree thank you for sharing your experience on this.

-Have you tried HBSS instead of PBS? because it has less phosphate so we thought it could be better for immunizing the animal and not perturbing their normal phosphate blood concentration that much. If yes, did you notice any difference between HBSS and PBS?

-Usually we add sodium azide fopr long term storage. Also do you have a reference on using it to presevrve the samples for short term and another reference mentioning that cells can stay preserved for up to one month at 4C?

-If you have a published reference about the cells fixation for the purpose of making antibodies please share it with me. Most of litterature discusses cell fixation for several immunoassays like Flow cytometry and IHC...but not for immunizing animals..

Thank you

Dhouha Daassi Hi

Are these mammalian or bacterial cells or something else?

Here are two references: regarding cell preparation for immunization:

Article PFA-fixed Hsp60sp-loaded dendritic cells as a vaccine for th...

Article Monoclonal Antibody Production Against Vimentin by Whole Cel...

I thought you planned on using this for imaging or flow cytometry, nonetheless fixation will be with formaldehyde based solvents (PFA ).

PBS is recommended for storage and is not directly injected. Once the cells arrive at their destination, they can be washed in PBS and reconstituted in the immunization cocktail, likely containing contains serum or adjuvants and other carriers or components.

Hope this helps

Best

Aparajita

Probable best not to fix at all !

Grow cells in serum free medium to reduce antibody response to this material.

Wash the cells well in eg serum free medium /pbs, and then freeze them down at -20 or -70c. Ship to the antibody producer on dry ice and thaw just before use.

Depends on how you will treat your samples later for analysis. If you ship them fixed with formaldehyde, but later plan to do studies on native cells, e.g for sorting them by FACS, you'll run into big trouble, I'm afraid.

As you'll be paying your contractor a lot of money for their services, they should be able to consult you in finding the optimal solution.

Dhouha Daassi in that case I don't see why you would want to fix the cells at all. I agree with Peter's suggestion and you can even refer to the second article sent by Aparajita. Fixation is necessary only for certain microscopy, flow or other marker based analyses.

And hope you are aware that some of the protocols shared above are for analysis and not for cell preparation for immunization.