I have supernatant from LPS stimulated splenocytes and lymph node cells.
Originally I planned on using ELISA to measure specific cytokines, however the antibodies I've been using (from mouse, rat, human) are proving ineffective at detecting any antigen.
Does anyone have any experience measuring immune function (outside of RNA measures) in Siberian hamsters, any ELISA to recommend that works in this species?
I have no experiance with siberian hamster cell cultures but Professor Stephan Steinlechner at the Hannover Veterinary School or his coworker Frank Scherbarth may have.
What you may also do is to make an extract of your LPS-stimulated cells and look for p42/44 MAPK or p38 phosphorylation, since this reaction is oberserved in many LPS stimulated cell systems (see Motzkus and Schulz-Maronde et al., FASEBJ, 2006). Hope this helps!
I have no experiance with siberian hamster cell cultures but Professor Stephan Steinlechner at the Hannover Veterinary School or his coworker Frank Scherbarth may have.
What you may also do is to make an extract of your LPS-stimulated cells and look for p42/44 MAPK or p38 phosphorylation, since this reaction is oberserved in many LPS stimulated cell systems (see Motzkus and Schulz-Maronde et al., FASEBJ, 2006). Hope this helps!
dear Jeremy
sometimes cell based assays work with mediators from other species. However, it is much more work than just an ELISA. The idea provided by Erik may also work.
You can compare the count of cells with B cell specific markers from LPS activated and none activated groups. Markers like CD19 or CD21 via Flow cytometry, or ELISA.
http://www.antibodyresource.com/search/Antibodies/97d45c5f-7a57-c6f6-c950-930011088ffd/cd19/reactivity/Hamster
Also you can use MTT assay and compare the cellular activity of LPS activated and none groups. However this test is not specific for B cells and will measure the activity of all cells in spleen like T cells.
Hi Jeremy,
You can try The reverse transcriptase polymerase chain reaction (RT-PCR) by extracting mRNA of spleen or LN cells (2 x 107 cells/mL) .
Fazzani's group tried it and you can find the paper through this link
http://www.scielo.br/pdf/bjmbr/v44n7/654.pdf
Hi All,
Thank you for all of your answers! I no longer have cells unfortunately, only the LPS stimulated supernate, so I have nothing to get mRNA from to look at gene expression. I checked how much protein I actually have, around 250-300 ug/mL, which is relatively low. Perhaps I need to concentrate the samples somehow (maybe trichloroacetic acid precipitation) and the ELISAs may work but the signal is below the standard curve. I am trying a western blot to see if I can get a band at the correct size of IL-1beta (~17.5 kDa) with some of the antibodies I have.
Hi Jeremy,
TNF-alpha may also work with supernatant and Western blot detection. Try first unchanged before you precipitate. Around 1 ng/ band may be enough, if the antibody works.
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