Purification and Immunophenotyping of BAL Cells

BAL cells were purified by centrifugation on Percoll to remove surfactant and debris ^{31 (link)}. BAL cells or BMDMs were immunostained to detect CD115 (AFS98), F4/80 (BM8) (eBioscience), CD3 (145-2C11), CD11b (M1/70), CD11c HL3), CD16/32 (2.4G2), CD19 (1D3), CD64 (X54-5/7.1.1), CD131 (JORO50), Ly6G (1A8), CD45.1 (A20), CD45.2 (104), MHC class II (I-A/I-E) (M5/114.15.2), SiglecF (E50-2440) (BD Biosciences), CD14 (Sa14-2) (BioLegend), CD68 (FA-11) (AbD Serotec), and MerTK (108928) (R&D Systems), as previously described^{1 (link)}, evaluated by flow cytometry using a FACSCaliber or FACSCanto flow cytometers (both from BD Biosciences, San Jose, CA), and the results were analyzed using CellQuest, FACSDiva (BD Biosciences), or FlowJo software (Tree Star). For intracellular staining of CD68, Leucoperm (AbDSerotec) was used as directed by the manufacturer.

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Suzuki T., Arumugam P., Sakagami T., Lachmann N., Chalk C., Sallese A., Abe S., Trapnell C., Carey B., Moritz T., Malik P., Lutzko C., Wood R.E, & Trapnell B.C. (2014). Pulmonary Macrophage Transplantation Therapy. Nature, 514(7523), 450-454.

Publication 2014

F4/80 (BM8) SiglecF (E50-2440) CD14 (Sa14-2) CD68 (FA-11) MerTK (108928) FACSCaliber FACSCanto CellQuest FACSDiva Leucoperm

Cd11b Cd131 Cells Centrifugation Flow cytometry Intracellular Mertk Mhc class ii Percoll Surfactant Tree

Corresponding Organization : Cincinnati Children's Hospital Medical Center

Other organizations : Medizinische Hochschule Hannover, Harvard University

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Variable analysis

independent variables

Centrifugation on Percoll to remove surfactant and debris

dependent variables

CD115 (AFS98), F4/80 (BM8), CD3 (145-2C11), CD11b (M1/70), CD11c HL3), CD16/32 (2.4G2), CD19 (1D3), CD64 (X54-5/7.1.1), CD131 (JORO50), Ly6G (1A8), CD45.1 (A20), CD45.2 (104), MHC class II (I-A/I-E) (M5/114.15.2), SiglecF (E50-2440), CD14 (Sa14-2), CD68 (FA-11), and MerTK (108928) expression on BAL cells or BMDMs

control variables

Not explicitly mentioned

positive controls

Not specified

negative controls

Not specified

Annotations

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