Isolation of Lung Immune Cells

To obtain single-cell suspensions from lung tissue, lungs were perfused with sterile PBS and removed en bloc, and perfused lungs were digested in RPMI medium containing collagenase XI (0.7 mg/mL; Sigma-Aldrich) and type IV bovine pancreatic DNase (30 μg/mL; Sigma-Aldrich). RBCs were lysed with RBC Lysis Buffer (BioLegend) as described elsewhere (62 (link)). Single-cell suspensions were incubated with a Fc receptor block (553141, BD Bioscience) to reduce nonspecific antibody binding. The flow cytometry panel used to identify immune cell subtypes is shown in Supplemental Figure 3; in short, antibodies used in these experiments included CD45-Brilliant Violet 650 (catalog 103151), Ly6G-APC/Cyanine7 (catalog 127623), F4/80-PE/Cy5 (catalog 123111), MerTK-Brilliant Violet 605 (catalog 151517), CD11c-AF-700 (catalog 117320), CD11b- PE/Cyanine7 (Cat.101216), MHCII-FITC (catalog 107605), and CD64-APC (catalog 139306) from BioLegend as well as SiglecF-PE (catalog 552126) from BD Biosciences. Dead cells were excluded using DAPI (catalog MBD0015, MilliporeSigma). Flow cytometry was performed using BD LSR II and BD FACS Aria III flow cytometers (BD Biosciences), and data were analyzed with FlowJo software.

Free full text: Click here

DiGiovanni G.T., Han W., Sherrill T.P., Taylor C.J., Nichols D.S., Geis N.M., Singha U.K., Calvi C.L., McCall A.S., Dixon M.M., Liu Y., Jang J.H., Gutor S.S., Polosukhin V.V., Blackwell T.S., Kropski J.A, & Gokey J.J. (2023). Epithelial Yap/Taz are required for functional alveolar regeneration following acute lung injury. JCI Insight, 8(19), e173374.

Publication 2023

collagenase XI type IV bovine pancreatic DNase RBC Lysis Buffer Fc receptor block CD45-Brilliant Violet 650 Ly6G-APC/Cyanine7 F4/80-PE/Cy5 MerTK-Brilliant Violet 605 CD11c-AF-700 CD11b- PE/Cyanine7 MHCII-FITC CD64-APC SiglecF-PE DAPI BD LSR II BD FACS Aria III

Antibodies Antibody Aria Bovine Buffer Cd11b Cells Collagenase Dapi Fc receptor Fitc Flow cytometry Lungs Mertk Pancreatic dnase Sterile Tissue Violet

Corresponding Organization : Vanderbilt University Medical Center

Other organizations : Vanderbilt University

Top 5 similar protocols

Variable analysis

independent variables

Collagenase XI (0.7 mg/mL)
Type IV bovine pancreatic DNase (30 μg/mL)

dependent variables

Immune cell subtypes identified by flow cytometry panel

control variables

Perfusion of lungs with sterile PBS
Lysis of RBCs with RBC Lysis Buffer
Incubation with Fc receptor block (553141, BD Bioscience) to reduce nonspecific antibody binding

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!