Preparation of Single Cell Suspension from Human Lung Tissue v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Peter A. Szabo ◽  
Basak Ural ◽  
Maya M.L. Poon
Keyword(s):  
Epithelial Cells ◽  
Cell Suspension ◽  
Single Cell ◽  
Lung Tissue ◽  
Immune Cells ◽  
Human Lung ◽  
Dilution Factor ◽  
Single Cell Suspension ◽  
Human Lung Tissue ◽  
Defined Media

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from human lung sections of about two grams. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of a Single Cell Suspension from Bronchoalevolar Lavage v1

2021 ◽  
Author(s):  
Peter A. Szabo ◽  
Steven B. Wells ◽  
Basak Ural
Keyword(s):  
Epithelial Cells ◽  
Cell Suspension ◽  
Single Cell ◽  
Immune Cells ◽  
Human Lung ◽  
Lavage Fluid ◽  
Cell Suspensions ◽  
Dilution Factor ◽  
Single Cell Suspension ◽  
Defined Media

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from lavage fluid collected from human lung. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Lymph Node Tissue v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Peter A. Szabo ◽  
Nora Lam ◽  
Maya M.L. Poon
Keyword(s):  
Lymph Node ◽  
Cell Suspension ◽  
Single Cell ◽  
Myeloid Cells ◽  
Cell Suspensions ◽  
Dilution Factor ◽  
Single Cell Suspension ◽  
Density Centrifugation ◽  
Defined Media ◽  
Human Lymph Node

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human lymph node tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Spleen Tissue v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Peter A. Szabo
Keyword(s):  
Cell Suspension ◽  
Single Cell ◽  
Myeloid Cells ◽  
Cell Suspensions ◽  
Dilution Factor ◽  
Single Cell Suspension ◽  
Human Spleen ◽  
Spleen Tissue ◽  
Density Centrifugation ◽  
Defined Media

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human spleen tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

scholarly journals Characterization of Early Stages of Human Alveolar Infection by the Q Fever AgentCoxiella burnetii

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Amanda L. Dragan ◽  
Richard C. Kurten ◽  
Daniel E. Voth
Keyword(s):  
Epithelial Cells ◽  
Lung Tissue ◽  
Alveolar Macrophages ◽  
Human Lung ◽  
Q Fever ◽  
Cell Types ◽  
Alveolar Epithelial Cells ◽  
Human Lung Tissue ◽  
Content Type ◽  
Alveolar Epithelial

ABSTRACTHuman Q fever is caused by the intracellular bacterial pathogenCoxiella burnetii. Q fever presents with acute flu-like and pulmonary symptoms or can progress to chronic, severe endocarditis. After human inhalation,C. burnetiiis engulfed by alveolar macrophages and transits through the phagolysosomal maturation pathway, resisting the acidic pH of lysosomes to form a parasitophorous vacuole (PV) in which to replicate. Previous studies showed thatC. burnetiireplicates efficiently in primary human alveolar macrophages (hAMs) inex vivohuman lung tissue. AlthoughC. burnetiireplicates in most cell typesin vitro, the pathogen does not grow in non-hAM cells of human lung tissue. In this study, we investigated the interaction betweenC. burnetiiand other pulmonary cell types apart from the lung environment.C. burnetiiformed a prototypical PV and replicated efficiently in human pulmonary fibroblasts and in airway, but not alveolar, epithelial cells. Atypical PV expansion in alveolar epithelial cells was attributed in part to defective recruitment of autophagy-related proteins. Further assessment of theC. burnetiigrowth niche showed that macrophages mounted a robust interleukin 8 (IL-8), neutrophil-attracting response toC. burnetiiand ultimately shifted to an M2-polarized phenotype characteristic of anti-inflammatory macrophages. Considering our findings together, this study provides further clarity on the uniqueC. burnetii-lung dynamic during early stages of human acute Q fever.

scholarly journals Optimization of Cryopreserved Single Cell Suspension of Gastric Epithelial Cells for Successful DotscanTM Antibody Microarray

2019 ◽  
Vol 48 (5) ◽  
pp. 1105-1110
Author(s):  
Asif Sukri ◽  
Alfizah Hanafiah ◽  
Nik Ritza Kosai ◽  
Mustafa Mohamed Taher ◽  
Isa Mohamed Rose ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Suspension ◽  
Single Cell ◽  
Gastric Epithelial Cells ◽  
Single Cell Suspension ◽  
Antibody Microarray

scholarly journals Cryobanking of human distal lung epithelial cells for preservation of their phenotypic and functional characteristics.

2021 ◽  
Author(s):  
Bindu Konda ◽  
Apoorva Mulay ◽  
Changfu Yao ◽  
Edo Israely ◽  
Stephen Beil ◽  
...  
Keyword(s):  
Single Cell ◽  
Lung Tissue ◽  
Human Lung ◽  
Alveolar Type ◽  
Basal Cells ◽  
Human Lung Tissue ◽  
Downstream Analysis ◽  
Long Term Preservation

The epithelium lining airspaces of the human lung is maintained by regional stem cells including basal cells of pseudostratified airways and alveolar type 2 pneumocytes (AT2) of the alveolar gas-exchange region. Despite effective methods for long-term preservation of airway basal cells, methods for efficient preservation of functional epithelial cell types of the distal gas-exchange region are lacking. Here we detail a method for cryobanking of epithelial cells from either mouse or human lung tissue for preservation of their phenotypic and functional characteristics. Flow cytometric profiling, epithelial organoid-forming efficiency, and single cell transcriptomic analysis, were used to compare cells recovered from cryopreserved tissue with those of freshly dissociated tissue. Alveolar type 2 cells within single cell suspensions of enzymatically digested cryobanked distal lung tissue retained expression of the pan-epithelial marker CD326 and the AT2 cell surface antigen recognized by monoclonal antibody HTII-280, allowing antibody-mediated enrichment and downstream analysis. Isolated AT2 cells from cryobanked tissue were comparable with those of freshly dissociated tissue both in their single cell transcriptome and their capacity for in vitro organoid formation in 3D cultures. We conclude that the cryobanking method described herein allows long-term preservation of distal human lung tissue for downstream analysis of lung cell function and molecular phenotype, and is ideally suited for creation of an easily accessible tissue resource for the research community.

Related expression of arachidonate 12- and 15-lipoxygenases in animal and human lung tissue

1991 ◽  
Vol 261 (6) ◽  
pp. L399-L405 ◽  
Author(s):  
V. R. Shannon ◽  
E. C. Crouch ◽  
Y. Takahashi ◽  
N. Ueda ◽  
S. Yamamoto ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Lung Tissue ◽  
Human Lung ◽  
Mononuclear Leukocytes ◽  
Human Lung Tissue ◽  
Intense Staining ◽  
Tracheal Epithelial Cells ◽  
Cell Extracts ◽  
Tracheal Epithelial ◽  
12 Lipoxygenase

We examined the immunohistochemical distribution of the arachidonate 12- and 15-lipoxygenases in anima l and human lung tissue using a polyclonal anti-12/15-lipoxygenase antibody. Immunoblotting of whole cell extracts fro m bovine and human tracheal epithelial cells or from bovine leukocytes with the antibody (raised originally against pu rified porcine leukocyte 12-lipoxygenase) showed immunoperoxidase staining of a single protein band (Mr = 72,000), whi ch com igrated with purified bovine 12-lipoxygenase. The antibody also immunoprecipitated both 12- and 15-lipoxygenase activities from cytosolic fractions of bovine and human tracheal epithelial cells. Immunohistochemistry of formaldehyd e-fixed and paraffin-embedded bovine (and ovine and canine) trachea using the same polyclonal antibody and an indirect biotin-avidin-peroxidase detection system demonstrated specific staining of tracheal epithelium, polymorphonuclear and mononuclear leukocytes, and perineural cells. Less intense staining of submucosal glands and blood vessels was also ob served. Lung sections demonstrated that the level of lipoxygenase antigen decreased markedly by the level of the bronc hi and was absent in more distal airways. A similar pattern of immunostaining was found in human lung, except that air way smooth muscle was also weakly reactive, and polymorphonuclear (neutrophilic) leukocytes were unstained (in accorda nce with the low 12/15-lipoxygenase activity in this cell type). We conclude that animal and human epithelial 12/15-li poxygenases share enzymatic, antigenic, and regional distribution characteristics and may therefore possess a common f unction in the pulmonary airway.

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