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B cell Elispot on mouse splenocytes

Elispot plasmablasts mouse splenocytes

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#1 lofangi

lofangi

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Posted 05 December 2013 - 09:37 AM

Dears, I perform B cell Elispot on mouse splenocytes to measure the number of antigen-specific plasmablasts 7-8 days after immunization with a protein antigen. I need your help to understand why my Elispot wells are so dirty (see the attached figure) after development. I use a common protcol that here I summarize:

1- coating ON 4° with the antigen or control (non-related antigen, I use BSA)

2- Blocking, 3h RT with PBS+10%FCS

3- I plate splenocyets at 800 000 cells/well and perform serial dilutions

4- incubation 3h 37°

4- wash plate 3 times with PBS

5- wash plate 3 times with PBS+Tween 0.05%

6- secondary antibody and wash

7- HRP and wash

8- AEC Kit and development.

 

As you can observe in the picture, at the highest dilution of cells (800 000 cells/well) I have very dirty wells that do not allow me to count the spots. This dirty decrease with the number of cells and is not observed when I perform the same protocol to detect Bone Marrow Plasmacells and Memory B cell Elsipot. The dirty is observed also in BSA coated wells.

I think it's something related to the mouse spleen that I'm not able to wash away. Does anyone has some suggestion about why it happen or a possible solution (different washing buffers)? 

Thank you very much,

lofangi

Attached Thumbnails

  • Dirty well.png






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