Bacterial cells were grown in defined medium as above for 24 and 48 h. Cultures were centrifuged and bacterial pellets were resuspended in 1× PBS and corrected to an optical density measured at 600 nm (OD600) of 1.0. Bone marrow was isolated from C57BL/6 mice and grown in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) (PreProTech) for 7 d. Once matured, the BMDCs were scraped and plated in 96-well plates and allowed to adhere. After at least 3 h, BMDCs were treated with whole bacterial cells overnight. Additionally, BMDCs were treated with pure capsule preparation overnight and then, the following morning, were treated with lipopolysaccharide (LPS) for 1.5 h. TNF-α production was detected using ELISAs (ThermoFisher) as described previously (16 (link)).
Protocol detail
Bone Marrow Dendritic Cell Responses to Bacterial Cells
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Bacterial
Bone marrow
C57bl mice
Capsule
Cells
Elisas
Granulocyte macrophage colony stimulating factor
Lipopolysaccharide
Pellets
Tnf α
Corresponding Organization :
Other organizations : Harvard University, Massachusetts General Hospital, Broad Institute
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Variable analysis
independent variables
- Duration of bacterial cell growth (24 h and 48 h)
- Treatment of bone marrow-derived dendritic cells (BMDCs) with whole bacterial cells overnight
- Treatment of BMDCs with pure capsule preparation overnight followed by lipopolysaccharide (LPS) for 1.5 h
- Tumor necrosis factor-alpha (TNF-α) production detected using ELISAs
- Defined medium used for bacterial cell growth
- Optical density (OD600) of bacterial cell suspension corrected to 1.0
- Bone marrow isolated from C57BL/6 mice and cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days to generate BMDCs
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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