Bone Marrow-Derived Macrophage Infection Protocol

Bone marrow-derived macrophages (BMDMs) from C57BL/6J (Jackson), Tnfrsf1a^−/− (23 (link)) (Jackson), Trif^−/− (24 (link)) and Trif^−/−Tnfr1^−/− mice (25 (link)) were cultured and infected as previously described (19 (link)). Y. pseudotuberculosis strain IP2666 and isogenic yopJ mutant bacteria were grown overnight with aeration in 2×YT broth at 26 °C. Yp were diluted into inducing media (2×YT containing 20mM sodium oxalate and 20mM MgCl₂) and grown with aeration for 1 h at 26 °C followed by 2 h at 37 °C. BMDMs were infected at a multiplicity of infection (MOI) of 20:1, unless otherwise noted. Cells were incubated at 37 °C and gentamicin (100 μg/mL) was added 1 h after infection. 100 μM zVAD-fmk (zVAD; SM Biochemicals), 60 μM necrostatin-1 (Nec-1; Calbiochem), 3 μM GSK2399872A (GSK’872; GlaxoSmithKline), 50μM TAPI-2 (Sigma), 80μM dynasore (Sigma) were added 1 h before infection where indicated.

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Peterson L.W., Philip N.H., Dillon C.P., Bertin J., Gough P.J., Green D.R, & Brodsky I.E. (2016). Cell-extrinsic TNF collaborates with TRIF signaling to promote Yersinia-induced apoptosis. Journal of immunology (Baltimore, Md. : 1950), 197(10), 4110-4117.

Publication 2016

C57BL/6J Tnfrsf1a−/− TAPI-2 dynasore

Bacteria Bone marrow derived macrophages Cells Dynasore Gentamicin Infection Mgcl2 Mice Nec 1 Necrostatin 1 Pseudotuberculosis Sodium oxalate Strain Tapi 2 Tnfr1 Zvad fmk

Corresponding Organization : University of Pennsylvania

Other organizations : St. Jude Children's Research Hospital, GlaxoSmithKline (United States)

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Variable analysis

independent variables

Mouse genotypes: C57BL/6J (Jackson), Tnfrsf1a−/− (23), Trif−/− (24), and Trif−/−Tnfr1−/− (25)
Bacterial strains: Y. pseudotuberculosis strain IP2666 and isogenic yopJ mutant

dependent variables

Infection outcome in bone marrow-derived macrophages (BMDMs)

control variables

Culturing and infection conditions of BMDMs, including multiplicity of infection (MOI) of 20:1, addition of gentamicin, and incubation temperature of 37°C
Bacterial growth conditions, including overnight culture in 2×YT broth at 26°C, followed by induction in media containing sodium oxalate and MgCl2 for 1 hour at 26°C and 2 hours at 37°C
Pharmacological inhibitors: 100 μM zVAD-fmk (zVAD), 60 μM necrostatin-1 (Nec-1), 3 μM GSK2399872A (GSK'872), 50 μM TAPI-2, 80 μM dynasore

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