CX3CR1 gene expression levels in peripheral blood mononuclear cells (PBMCs) related to alpha-1 antitrypsin-deficiency (AATD) and plasma concentrations of CX3CR1 ligand (CX3CL1) and Z-AAT polymer. (A)…
Source files, containing original data for Figure 1A and B, to document CX3CR1 expression (A), and plasma levels of CX3CL1 in alpha-1 antitrypsin-deficient (AATD) and non-AATD individuals (B).
The German cohort comprises 41 ZZ alpha-1 antitrypsin-deficient (AATD) volunteers irrespective of clinical status and medications. For control, we collected blood from 21 non-AATD volunteers with …
(A) CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 …
Source file, containing original data for Figure 2A, to document reduced CX3CR1 expression in peripheral blood mononuclear cells (PBMCs) treated with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) (A).
(A) Plasma M- and Z-AAT was purified by affinity chromatography with AAT-specific Alpha-1 Antitrypsin Select matrix. Proteins were run on a 10% SDS-PAGE gel and stained with Coomassie Blue G250. AAT …
PBMCs were incubated for 18 hr with plasma-derived Z-AAT in the concentrations as indicated. CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman …
Lipid rafts were solubilized from membrane fractions with UltraRIPA kit. (a) For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. One representative …
(A) CX3CR1 mRNA expression relative to HPRT1 was determined by real-time PCR using Taqman gene expression assays. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr in RPMI medium …
Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr with CX3CL1 and Z-AAT polymers separately or in combination or with RPMI medium alone (control). (A) CX3CR1 gene expression …
Source file, containing original data for Figure 2—figure supplement 5A, to document CX3CR1 gene expression in peripheral blood mononuclear cell (PBMC).
CX3CR1-expressing cells were found in the monocyte gate (A) and in the NK cell gate (CD56lo CD16+) (B). Histograms show representative results and bars represent mean (SD) of n = 4 independent …
Source files, containing original data for Figure 3A,B, to document reduced CX3CR1 surface expression in monocytes (A) and NK cells (B) after treatment with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) (A).
PBMCs were stained with CD14, CD16, and CD56 in the presence of 7-amino-actinomycin D (7-AAD). Dead cells were excluded by 7-AAD. From the single cells, CD14+ cells with a mid-level of granularity …
(A) Z-AAT increases CD14 gene expression. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr with 0.5 mg/ml Z-AAT or RPMI medium alone (control). CD14 mRNA expression relative to …
Source files, containing original data for Figure 4A, B to document CD14 gene expression in peripheral blood mononuclear cells (PBMCs) (A) and CD14 surface expression in monocytes (B).
PBMCs were incubated for 18 hr with plasma-derived Z-AAT in the concentrations as indicated, or with RPMI medium alone (control). CX3CR1 and CD14 gene expression relative to HPRT1 housekeeping gene …
Peripheral blood mononuclear cells (PBMCs) were treated with Z-AAT or lipopolysaccharide (LPS) (used as a positive control) for 18 hr. IL1B (a), IL6 (b), and TNFA (c) mRNA expression relative to HPRT…
Source files, containing original data for Figure 4—figure supplement 2 to document IL1B (a), IL6 (b), and TNFA (c) gene expression in peripheral blood mononuclear cells (PBMCs).
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Biological sample (Homo sapiens) | PBMCs | Blood samples | Collected from 41 ZZ AATD and 21 non-AATD healthy controls | |
Sequenced-based reagent (human) | CX3CR1 | Thermo Fisher Scientific | Taqman assay 4331182 | Hs00365842_m1 |
Sequenced-based reagent (human) | TNFA | Thermo Fisher Scientific | Taqman assay 4331182 | Hs01113624_g1 |
Sequenced-based reagent (human) | IL6 | Thermo Fisher Scientific | Taqman assay 4331182 | Hs00985639_m1 |
Sequenced-based reagent (human) | IL1B | Thermo Fisher Scientific | Taqman assay 4331182 | Hs01555410_m1 |
Sequenced-based reagent (human) | HPRT1 | Thermo Fisher Scientific | Taqman assay 4331182 | Hs02800695_m1 |
Sequenced-based reagent (human) | CD14 | Thermo Fisher Scientific | Taqman assay 4331182 | Hs00169122_g1 |
Other | Non-adherent 12-well plates | Greiner Bio-One | 665970 | |
Other | Alpha-1 Antitrypsin Select matrix | GE Healthcare Life Sciences, Cytiva | 17547201 | |
Other | Plasma purified human AAT | CSL Behring | Respreeza | |
Other | LPS | Sigma-Aldrich | L2880 | Escherichia coli O55:B5 |
Commercial assay or kit | Pierce Chromogenic Endotoxin Quant Kit | Thermo Fisher Scientific | A39553 | |
Commercial assay or kit | UltraRIPA kit | BioDynamics Laboratory | F015 | |
Antibody | Anti-AAT polymer, LG96 (mouse monoclonal) | Deposited at German Collection of Microorganisms and Cell Cultures: DSM ACC3092 | LG96 | ELISA: Coating: 1 µg/ml; LG96-HRP conjugate: (1:2000) Immunofluorescence: (1:5000) |
Antibody | Anti-human AAT (rabbit polyclonal) | Agilent Dako | A001202-2 | (1:800) |
Antibody | Anti-AAT polymer (mouse monoclonal) | Hycult Biotech | Clone 2C1; HM2289 | (1:500) |
Antibody | Anti-CX3CR1 (rabbit polyclonal) | Abcam | ab8021 | (1:500) |
Antibody | Anti-β-actin (mouse monoclonal) | Sigma-Aldrich | Clone AC-15; A3854 | HRP-conjugated; (1:20,000) |
Antibody | Anti-CX3CR1 (rabbit polyclonal) | Abcam | ab8021 | (1:500) |
Antibody | Anti-CX3CR1 (mouse monoclonal) | Invitrogen, Thermo Fisher Scientific | Clone 2A9-1; 12-6099-42 | PE-conjugated; 5 µl per test |
Antibody | Anti-CD14 (mouse monoclonal) | Invitrogen, Thermo Fisher Scientific | Clone TuK4; MHCD1401 | FITC-conjugated; 5 µl per test |
Antibody | Anti-CD16 (mouse monoclonal) | Immunotools | Clone 3G8; 21278166 | APC-conjugated; 5 µl per test |
Antibody | Anti-CD56 (mouse monoclonal) | BD Biosciences | Clone NCAM16.2; 566124 | BV-480 conjugated; 5 µl per test |
Peptide, recombinant protein | Human CX3CL1/Fractalkine | Bio-Techne | 365-FR-025 | |
Software, algorithm | FlowJo | Becton, Dickinson and Company | v10 | |
Software, algorithm | SigmaPlot 14 | Systat Software | v14.0 |
Primer | Assay ID |
---|---|
CX3CR1 | Hs00365842_m1 |
TNFA | Hs01113624_m1 |
IL6 | Hs00985639_m1 |
IL1B | Hs01555410_s1 |
HPRT1 | Hs02800695_m1 |
CD14 | Hs00169122_g1 |