I incubated the spleen cells isolated from C57BL mice in RPMI 1640 medium for 24 hours to acclimate to the medium. I then exposed my cells to LPS at different concentrations for 24 hours (0.1ug 1 ug and 2 ug). I also incubated my cells with LPS as a treatment at the same time as a live, CFS, and heat-killed bacterium. At the end of the incubation, I collected my supernatants and cleared my samples from cells and bacteria by centrifugation and stored my samples at -80 degrees.

However, there was no cytokine secretion in the ELISA assay (TNF-a and IFN-gamma)

I got positive results in cell viability and cell proliferation.

LPS increased cytokine release in RAW 264.7 cells specifically (TNF-a, IL-10 and IL1-B)

While my cells are alive and the LPS I use stimulates cytokine release in different cell lines, why doesn't it stimulate cytokine release in mouse spleen cells?

My mouse isolation protocol and references I use:

http://www.kpubs.org/article/articleMain.kpubs?articleANo=E1MBA4_2016_v26n3_469

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