Table 1.
Characteristics of antibodies used in experiments.
Fig 1.
Overall approach to the isolation and phenotyping of rat pulmonary microvascular endothelial cells (PMVECs).
(A) Flow diagram of the initial strategy for presumed rat PMVEC isolation and phenotyping by immunocytochemistry and immunofluorescence. (B) Approach to isolating and confirming rat PMVECs acquired ex vivo by fluorescence activated cell sorting. The figure corresponding to data for a step in the approach is provided. HLF, human lung fibroblast; HPAEC, human pulmonary artery endothelial cell; HPASMC, human pulmonary artery smooth muscle cell; MCT, monocrotaline; RLF, rat lung fibroblast; RPAEC, rat pulmonary artery endothelial cell; RPASMC, rat pulmonary artery smooth muscle cell; rat PMVEC, rat pulmonary microvascular endothelial cell; SD, Sprague Dawley.
Fig 2.
Immunocytochemistry and immunofluorescence were unsuccessful for definitively identifying presumed pulmonary microvascular endothelial cells (PMVECs) isolated from rats ex vivo.
(A) Peripheral rat lung tissue underwent mechanical and enzymatic dissociation, and the cell pellet was cultured in endothelial-selective medium. Presumed rat PMVECs were analyzed using anti-CD31 Abs #1 and #2 immunocytochemistry (ICC). Luminosity was normalized to IgG control. (B) Anti-CD31 Ab #1, anti-von Willebrand factor (vWF) Ab #6, and anti-smooth muscle actin (α-SMA) Ab #7 ICC performed in presumed rat PMVECs, human lung fibroblasts (HLFs) and human pulmonary artery smooth muscle cells (HPASMCs) shows no significant difference in signal intensity by cell marker across different cell types. (C) Presumed rat PMVECs and HPAECs, HPASMCs, as well as HLFs as controls were analyzed by immunofluorescence (IF). Only CD31, and not vWF, signal was increased in presumed rat PMVECs compared to non-endothelial controls. Representative photomicrographs shown. a.u., arbitrary units. Student’s unpaired t-test or ANOVA. Means ± SE, N = 3-5/condition.
Fig 3.
Identifying human pulmonary artery endothelial cells (HPAECs) by flow cytometry.
(A) Commercially purchased HPAECs were analyzed by flow cytometry using anti-CD31 and anti-CD144 Abs #12 and #13. Compared with HPASMCs and HLFs, high expression of CD31 and CD144 was observed only in HPAECs. These results served as a positive control for further experiments aiming to confirm that cells isolated from rat lungs ex vivo were, in fact, endothelial. (B) Commercially purchased HPAECs were used to test the generalizability of these results. Alternative anti-CD31 and anti-CD144 antibodies did not reliably identify endothelial cell surface markers, supporting our earlier findings indicating variability in reactivity (i.e., quality) of tested antibodies across experimental methods, including flow cytometry. Representative plots and histograms shown. Means ± standard error, % CD31 or CD144 positive, N = 4-5/condition. Ab, antibody; Iso, Isotype control.
Fig 4.
Identifying rat pulmonary endothelial cells by flow cytometry.
Presumed rat PMVECs isolated by mechanical and enzymatic dissociation of peripheral lung and culture in endothelial-selective medium, commercial rat pulmonary artery endothelial cells (RPAECs), or rat pulmonary artery smooth muscle cells (RPASMCs) were labeled with antibodies against endothelial surface markers. (A) Anti-CD31 (Ab #12 and #16) and CD144 (Ab #13) signal was not observed in presumed rat PMVECs by flow cytometry (N = 3/condition). (B) Labeling of RPAECs was also not observed for anti-CD31 antibodies #12 (N = 4/condition) and (C) #16, respectively (N = 3/condition). (D) False-positive signal was detected in RPASMCs labeled with anti-CD144 Ab #19 (N = 4/condition). Representative plots and histograms shown. Means ± standard error, % CD31 or CD144 positive. Ab, antibody; Iso, Isotype control.
Fig 5.
Fluorescence-activated cell sorting (FACS) permits isolation of rat pulmonary microvascular endothelial cells (PMVECs).
Presumed rat PMVECs isolated from peripheral lung by immunomagnetic anti-CD31 bead selection, as well as commercial rat pulmonary artery endothelial cells (RPAECs), rat lung fibroblasts (RLFs) and RPASMCs were analyzed by flow cytometry directed against endothelial surface markers. (A) Anti-CD31 Ab #20 selectively labeled RPAECs relative to isotype control and RPASMCs (N = 4/condition). (B) Presumed rat PMVECs demonstrate specific anti-CD31 labeling relative to commercial rat lung fibroblasts (RLFs) and RPASMCs (N = 3/condition). (C) Presumed rat PMVECs also demonstrate specific signal for isolectin 1-B4 from Griffonia simplicifolia (GS-IB4) relative to RLFs and RPASMCs (N = 4/condition). (D) Over 90% of CD31-positive presumed rat PMVECs co-label with GS-IB4 (N = 3/condition). Confirmed rat PMVECs were defined as those cells positive for both CD31 and GS-IB4 by flow cytometry. Representative plots and histograms shown. Means, % CD31 or GS-IB4 positive. Ab, antibody; Iso, Isotype control.
Table 2.
Assessment of RNA quality by RNA integrity number.
Fig 6.
Reagent, personnel, and equipment costs attributable to the validation of commercial biomaterials.
Distribution of expenses attributable to the validation of commercial products used in the isolation of rat pulmonary microvascular endothelial cells. FACS, fluorescence-activated cell sorting.