Journal: Cell

Article Title: Lymph Nodes are Innervated by a Unique Population of Sensory Neurons with Immunomodulatory Potential

doi: 10.1016/j.cell.2020.11.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Next, using the Miltenyi CD3ε microbead kit and CD19 mouse microbead kit, all remaining LN cells were stained according to manufacturer instructions with the following modifications.

Techniques: Virus, Recombinant, Multiplex Assay, Expressing, Software

Journal: Cell

Article Title: Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae

doi: 10.1016/j.cell.2023.05.008

Figure Lengend Snippet: Colonization and death of macrophages differentiated from CD14 + monocytes isolated from human blood by V. cholerae , and attachment of bacteria to primary CD14 + monocytes, refers to Figures 1 , , , and (A) V. cholerae WT exhibits strongly reduced attachment to CD14 + monocytes compared with macrophages (Mφ) derived from CD14 + monocytes. Attachment of V. cholerae to immune cells was quantified from confocal images after 0.5 h of co-incubation and normalized to the mean value for macrophages. Statistical significance was calculated using an unpaired t test (number of independent biological replicates: n macrophage = 12, n monocyte = 3; ∗∗∗∗ indicates p < 0.0001). Error bars denote the standard deviation. (B) Attachment capabilities of different V. cholerae strains to the surface of macrophages derived from CD14 + monocytes normalized to the mean value of WT bacteria. For bacteria with a stalled flagellar motor (achieved by the addition of phenamil), cells that lack the polar flagellum (Δ flaA ), or cells lacking MSHA pili (Δ mshA ), attachment to macrophages is attenuated. Ectopic expression of mshA or flaA under control of the native promoter restores bacterial attachment. Statistical significance was calculated using one-way ANOVA (number of independent biological replicates: n = 3–16; ∗ indicates p < 0.05; ∗∗ indicates p < 0.005; ∗∗∗∗ indicates p < 0.0001). Error bars denote the standard deviation. (C) Representative microscopy images from n = 3–8 independent biological replicates show V. cholerae biofilms (cyan) formed on primary macrophages (red) differentiated from CD14 + monocytes isolated from human blood, imaged at peak time of biofilm formation. The V. cholerae biofilm matrix components Bap1, RbmA, RbmC, and Vibrio polysaccharide (VPS) are not required for biofilm formation on human macrophages. Instead, V. cholerae biofilm formation on macrophages derived from CD14 + monocytes depends on the production of MSHA pili and TC pili (with the secreted protein TcpF). (D) During co-incubation of macrophages with V. cholerae lacking hemolysin HlyA, macrophage death is significantly reduced compared with WT bacteria. Overexpression of hlyA under control of the inducible P tac promoter restores and enhances bacteria-induced killing of macrophages. Macrophage death was measured after 7 h of exposure to bacteria. Bars represent the percentage of dead macrophages for different V. cholerae strains, normalized to the WT mean value. Statistical significance was calculated using an unpaired t test (n = 3–11 independent biological replicates; ∗∗∗∗ indicates p < 0.0001). Error bars denote the standard deviation. (E) Biofilm growth on macrophages supports hemolysin-dependent death of macrophages. During co-incubation, V. cholerae strains deficient in the formation of biofilms on macrophages (strains carrying the Δ mshA and Δ tcpA mutations) cause less death of macrophages compared with biofilm-capable bacteria. Bars represent the percentage of dead macrophages for different V. cholerae strains normalized to the WT. Macrophage death was measured after 7 h of exposure to bacteria. Statistical significance was calculated using an unpaired t test (n = 3–11 independent biological replicates; ∗∗∗∗ indicates p < 0.0001; ∗∗ indicates p < 0.007). Error bars denote the standard deviation.

Article Snippet: The leukocytes were then isolated using the Lymphoprep density gradient medium (STEMCELL Technologies cat. no. 07801) and were further separated into monocytes, CD4 + T cells, B cells, as well as NK cells with the help of Milteny MACS MicroBeads (CD14, CD4, CD19, CD56) according to the manufacturer’s protocol.

Techniques: Isolation, Derivative Assay, Incubation, Standard Deviation, Expressing, Microscopy, Over Expression

Journal: Cell

Article Title: Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae

doi: 10.1016/j.cell.2023.05.008

Figure Lengend Snippet:

Article Snippet: The leukocytes were then isolated using the Lymphoprep density gradient medium (STEMCELL Technologies cat. no. 07801) and were further separated into monocytes, CD4 + T cells, B cells, as well as NK cells with the help of Milteny MACS MicroBeads (CD14, CD4, CD19, CD56) according to the manufacturer’s protocol.

Techniques: Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Mass Spectrometry, Software