Multiparametric Flow Cytometry of Lung Immune Cells

Cells in BALF and lung homogenate were stained with fluorescently labeled antibodies: CD11b-AF594 (M1/70), Ly6G-BV605 (1A8), CD11c-PerCP-Cy5.5 (N418), CD45-AF700 (30-F11), BV510-CD103 (2E7), Siglec-F-AF647 (E50–2440; BD Biosciences), MHCII-APC-Cy7 (M5/114.15.2), Ly6C-PE-Texas Red (AL-2; BD Biosciences) and DAPI to assess cell viability. Antibodies were from Biolegend unless otherwise stated. Cells in the airway and lung were classified as before (40 (link), 41 (link)). For IL-1β intracellular staining, surface staining was performed as stated above and was followed by fixation and then permeabilization, using the fluorescently labeled antibody pro-IL-1β-APC (NJTEN3) and the two-step protocol for intracellular (cytoplasmic) proteins (eBioscience) according to manufacturers instructions. We have adhered to the EJI recommended guidelines for the use of flow cytometry.

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Pires S, & Parker D. (2018). Activation of IL-1β in response to Staphylococcus aureus in the lung requires inflammasome-dependent and independent mechanisms. European journal of immunology, 48(10), 1707-1716.

Publication 2018

Siglec-F-AF647 MHCII-APC-Cy7 (M5/114.15.2), Ly6C-PE-Texas Red (AL-2;

Af647 Antibodies Antibody Cd103 Cd11b Cell viability Cells Cy5 5 Cytoplasmic Dapi Flow cytometry Il 1β Intracellular Lung Proteins Siglec

Corresponding Organization : Columbia University

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Variable analysis

independent variables

Staining of cells in BALF and lung homogenate with fluorescently labeled antibodies: CD11b-AF594 (M1/70), Ly6G-BV605 (1A8), CD11c-PerCP-Cy5.5 (N418), CD45-AF700 (30-F11), BV510-CD103 (2E7), Siglec-F-AF647 (E50–2440), MHCII-APC-Cy7 (M5/114.15.2), Ly6C-PE-Texas Red (AL-2)

dependent variables

Cell populations in the airway and lung classified based on the antibody staining

control variables

DAPI to assess cell viability
Antibody isotype controls not explicitly mentioned

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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