Journal: International journal of molecular sciences

Article Title: Altered Serum Alpha1-Antitrypsin Protease Inhibition before and after Clinical Hematopoietic Stem Cell Transplantation: Association with Risk for Non-Relapse Mortality.

doi: 10.3390/ijms25010422

Figure Lengend Snippet: Figure 3. The effect of clinical-grade AAT on patient-derived myeloid dendritic cells. (A) Peripheral blood samples from aGVHD patients (n = 18) and GVHD-free (n = 17) patients. Percent conventional dendritic cells of type 1 (CD141+) and type 2 (CD1c+) out of CD14-CD11C+HLA-DR+ cells. (B–E) Whole blood and PBMC stimulation assays: samples from day +28 incubated with LPS (10 ng/mL) or R848 (100 ng/mL), ±AAT (0.5 mg/mL). (B) IL-6 and IL-10 supernatant levels. aGVHD (n = 17) and GVHD- free (n = 15–16). Two-way ANOVA with Sidák’s post hoc test. Mean ± SEM. (C,D) Membranal expression by flow cytometric analysis of B7-H4 and CD40 on cDC1 and cDC2 (n = 34). (E) Membranal expression of B7-H4 by flow cytometric analysis according to disease status: aGVHD (n = 17) or GVHD-free (n = 17), and (F) according to median cutoffs: median AAT concentration (1.68 mg/mL, n = 16, 18) or median PR3/AAT/CRP (29 AU, n = 17, 17). Two-way ANOVA with Sidák’s post hoc test. Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Blocking was performed prior to staining using Tandem Signal Enhancer (Miltenyi Biotec, cat. 130- 099-887, Bergisch Gladbach, Germany) at room temperature for 20 min, then cells were stained with antibodies purchased from Miltenyi Biotec for CD1c (clone REA694, cat. 130- 110-536), CD11c (clone REA618, cat. 130-114-584), CD14 (clone REA599, cat. 130-110-523), CD141 (clone REA674, cat. 130-110-928), CD127 (clone REA614, cat. 130-109-518), CD25 (clone REA570, cat. 130-109-076), CD3 (clone REA613, cat. 130-110-464), CD366-TIM3 (clone REA635, cat. 130-109-759), CD4 (clone REA623, cat. 130-110-950), CD40 (clone REA733, cat. 130-110-950), CD8 (clone REA734, cat. 130-110-820), FoxP3 (clone 3G3, cat. 130-093- 014), and HLA-DR (clone REA805, cat. 130-111-792).

Techniques: Derivative Assay, Incubation, Expressing, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 1. SCARF1 profiling in immune cells. (A) SCARF1 is highly expressed on endothelial cells. CRISPR-Cas9 deletion of SCARF1 was per- formed on the human endothelial cell line, TIME. The histogram is repre- sentative of three independent experiments. (B) Uptake of ACs in SCARF11 (sgControl) and SCARF1−(sgSCARF1) endothelial cells. Cells were stimulated with ACs for 30, 60, or 90 min. AC uptake was measured by flow cytometry. Results shown are the average percent uptake ± SD of two independent experiments performed in triplicates. p 5 0.03 by Student t test analysis. (C) SCARF1 is expressed on phagocytes. PBMCs were iso- lated from the blood of healthy donors (n 5 5) and stained for flow cytome- try against SCARF1 and immune cell markers. TIME cells were used as a positive control. Data represent the mean (± SEM) of two independent experiments with two to three replicates. (D) Representative flow cytometry plot of total SCARF1 expression in the presence or absence of ACs. PBMCs gated on CD451CD11c1BDCA11 DCs were incubated in the presence or absence of Texas Redlabeled ACs for 4 h. Intracellular and extracellular stains were performed for SCARF1 to measure the total amount of SCARF1 interacting with ACs. Data shown are representative of three independent experiments (n 5 3) with similar results. (E) SCARF1 is upregulated after stimulation with ACs. BDCA11 DCs (red), CD141

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: CRISPR, Flow Cytometry, Staining, Positive Control, Expressing, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 2. SCARF1 is a nonredundant efferocytosis receptor on BDCA11 DCs. (A) SCARF1 is internalized after interacting with ACs. PBMCs (1 × 106 cells/ml) from healthy control subjects (n 5 5) were stimulated with ACs (2 × 106 cells/ml) for 1 h. After 1 h, media were removed, and new media were added. Cells were incubated for 3 h, and PBMCs were surface stained for flow cytometry. Data represent the mean (± SEM) of three independent experiments with one to two independent donors. Percent reduction is shown on the graph. ***p < 0.001. (B) SCARF1 is a phagocytic receptor for ACs in conventional DCs. MUTZ-3 DC line confocal images of SCARF1 (green), DNA (blue, DAPI), and ACs (red) 2 h poststimulation. Representative images from two independent experi- ments with three replicates. Original magnification, ×40 (left and middle); ×20 (right). Arrows show the colocalization of ACs and SCARF1. (C and D) Blocking SCARF1 reduces AC uptake. PBMCs (1 × 106/ml) were incubated with anti-SCARF1 for 30 min and then stimulated with pHrodo redlabeled ACs (2 × 106/ml). Cells were incubated for 3 h and then stained and analyzed by flow cytometry against CD451CD11c1BDCA11 (BDCA11 DCs) or CD451CD11cintCD11b1CD141

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Control, Incubation, Staining, Flow Cytometry, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 3. SCARF1 is involved in the inflammatory response. (A) NanoString gene profiling of SCARF1lo and SCARF1hi BDCA11 DCs. PBMCs were isolated from healthy control subjects and treated with ACs for 4 h. Live BDCA11 DCs (CD11c1BDCA11Live/Dead-UV) were sorted into SCARF1 negative (SCARF1lo) or SCARF1 positive (SCARF1hi). mRNA was extracted, and genetic profiling was performed using a Nano- String immunology panel. Heatmap showing the top 35 differentially expressed genes (log 2) after normalization using Morpheus software. Rep- resentative heatmap of single NanoString assay (n 5 4). p values by Stu- dent t test. (BD) Expression of IL-10 on BDCA11 DCs. BDCA11 sorted DCs (1 × 106/ml) were blocked or not with anti-SCARF1 blocking Ab for 30 min; then they were stimulated with Live/Dead-labeled ACs (2 × 106/ ml) for 4 h. (B) Cells were stained and analyzed for flow cytometry CD11c1CD11b1BDCA11SCARF11 and intracellular stain for IL-10. Data represent the mean (± SEM) of three independent experiments. p < 0.002 by two-way ANOVA. (C) Cells were lysed, and mRNA was measured using qPCR. Data are the mean (± SEM) of two independent experiments with n 5 3. p 5 0.01 by MannWhitney U test. (D) Cells were lysed, and mRNA was measured using qPCR. Data are the mean (± SEM) of two independent experiments with n 5 5. p 5 0.003 by MannWhitney U test. MFI, mean fluorescent intensity; U, unstimulated.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Isolation, Control, Software, Expressing, Blocking Assay, Labeling, Staining, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 4. SCARF1 regulates AC- induced phosphorylation of MAPK and STAT kinases. (A) Upregulation of STAT genes in SCARF11 BDCA11 DCs. PBMCs were isolated from healthy control subjects and treated with ACs for 4 h. Live CD11c1BDCA11 DCs were sorted into SCARF1-negative (SCARF1lo) or SCARF1- positive (SCARF1hi) groups. mRNA was extracted, and the genetic profiling was mea- sured by NanoString immunology panel. Unbiased heatmap depicting the regulation of kinases. Gene expression as log2 after normalization using Morpheus software. Representative heatmap of single NanoString assay (n 5 4). p values by Student t test. (BI) SCARF1 is partly responsible for phosphorylation of kinases. PBMCs from healthy control subjects (1 × 106/ml) were treated with SCARF1-blocking Ab for 30 min or left untreated; then Live/Dead- UVlabeled ACs were added for 15 or 30 min. Cells were immediately fixed and stained for flow cytometry. Cells were gated on CD11c1BDCA11SCARF11 in the pres- ence or absence of ACs. Left panel, MFI quantification; right panel, representative his- tograms. (B and C) p-MAPK p38 Thr180/ Tyr182, (D and E) p-STAT3 Tyr705, (F and G) p-STAT1 Tyr701, (H and I) p-ERK p44/ 42 Thr202/Tyr204. Data represent the mean (± SEM) of two independent experiments; n 5 4 independent individuals. *p < 0.02 by the MannWhitney U test. MFI, mean fluorescent intensity; ns, not significant; U, unstimulated.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Phospho-proteomics, Isolation, Control, Gene Expression, Software, Blocking Assay, Staining, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 5. SCARF1 expression is dysre- gulated after AC uptake on BDCA11 DCs. (A) The basal expression of SCARF1 is comparable between patients with SLE and healthy control subjects. PBMCs were iso- lated from healthy control subjects (n 5 7) or patients with SLE (n 5 17). PBMCs were stained for SCARF1, BDCA11 DCs, plas- macytoid DCs, and monocytes. Data are shown as MFI of SCARF1 surface expres- sion. Data are not significant by Man- nWhitney U test. (B) Representative flow cytometry for healthy control subjects and patients with SLE. Scheme for selection of CD11c1BDCA11 cells. Histogram of basal SCARF1 expression on BDCA11 DCs. Gray: healthy; black: SLE. (CF) SCARF1 contributes to the removal of ACs in patients with lupus. (C and D) PBMCs (1 × 106/ml) were incubated with anti-SCARF1 for 30 min and then stimulated with pHrodo redlabeled ACs (2 × 106/ml). Cells were incubated for 4 h. Cells were stained and analyzed by flow cytometry for SCARF1 and BDCA11 DCs (C) or CD141 mono- cytes (D). Data are the mean (± SEM) of three independent experiments with n 5 3 per group. (E and F) BDCA11 sorted DCs or CD141 monocytes (1 × 106/ml) were incu- bated with anti-SCARF1 for 30 min and then stimulated with ACs (2 × 106/ml) for 4 h. Cells were lysed and mRNA levels of Scarf1 (E) or Il10 (F) were measured using qPCR. Data are the mean (± SEM) of three independent experiments. *p < 0.01, **p < 0.005 by two-way ANOVA with Geissler- Greenhouse correction; ***p < 0.0001 by two-way ANOVA. MFI, mean fluorescent intensity; ns, not significant.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Expressing, Control, Staining, Flow Cytometry, Selection, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 6. Increased levels of anti-SCARF1 autoantibodies in the sera of patients with lupus show defects in removal of AC. (AC) Soluble SCARF1 or blocking buffer-coated ELISA plates were incubated with sera from patients with SLE or healthy individuals. IgG SCARF1 Abs (healthy: n 5 100; SLE: n 5 145) (A), IgM SCARF1 Abs (healthy: n 5 9; SLE: n 5 62) (B), and IgA SCARF1 Abs (healthy: n 5 9; SLE: n 5 62) (C). Data are shown as anti- SCARF1 minus antiblock data to reduce the level of binding to the blocking buffer. The OD values of each individual are represented as a single point. Dashed lines indicate the OD values that exceed the mean controls by >3 SDs. Horizontal bars represent the mean values. p 5 0.0009, p 5 0.0003, by Man- nWhitney U test. (DF) Lupus serum reduces AC uptake in SCARF11BDCA11 DCs. PBMCs (1 × 106/ml) were incubated with autologous serum, lupus serum, or serum starved for 2024 h. Cells were incubated for 3 h with pHrodo redlabeled ACs (2 × 106/ml). Cells were stained and analyzed by flow cytometry. (D) Representative histogram of pHrodo expression in SCARF11 BDCA11 DCs. (E) Percentage of ACs phagocytosed by BDCA11 DCs treated with the specified serum and measured as pHrodo-positive cells. (F) MFI quantification of SCARF1 expression from BDCA11 DCs that have phagocytosed ACs. (E and F) Data represent the mean (± SEM) of three independent experiments; n 5 5, by two-way ANOVA. (G and H) Levels of autoantibodies to SCARF1 directly correlate with deficiency in efferocytosis. PBMCs (1 × 106/ml) were incubated with autologous serum, lupus serum, or serum starved for 2024 h. Cells were incubated for 3 h with pHrodo redlabeled ACs (2 × 106/ml). Cells were stained and analyzed by flow cytometry. (G) Representative histogram of pHrodo expression in SCARF11 BDCA11 DCs. (H) Percentage of pHrodo-labeled ACs phagocytosed by BDCA11 DCs treated with specified serum and measured as pHrodo-positive cells. Data represent the mean (± SEM) of two independent experiments; n 5 12, by two-way ANOVA. (I) Autoanti- bodies block SCARF1 expression on BDCA11 DCs. PBMCs (1 × 106/ml) were incubated with autologous serum, lupus serum, or serum starved for 2024 h. Cells were incubated for 3 h with pHrodo redlabeled ACs (2 × 106/ml). Cells were stained and analyzed by flow cytometry. Data represent the mean (± SEM) of two independent experiments; n 5 12, by two-way ANOVA. ns, not significant.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Staining, Flow Cytometry, Expressing, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 7. Ig depletion increases SCARF1-mediated efferocytosis. (A) Decreased SCARF1 binding after IgG depletion. We depleted 20% serum in RPMI, healthy or SLE, using 100 ml of protein A/G agarose beads columns. IgG depletion was confirmed by Western blot (data not shown). SCARF1 bind- ing was analyzed by dot blot. Recombinant protein was transferred to nitrocellulose membrane, and full or depleted serum was used as a primary Ab. Human IgG was used as secondary Ab. Representative blot (SLE: n 5 13; healthy: n 5 8). Anti-SCARF1 was used as a control. (BF) Ig depletion restores efferocy- tosis on BDCA11 DCs in SLE patient serum. PBMCs (1 × 106/ml) were incubated with full or Ig-depleted serum for 2024 h. FBS was used as serum con- trol. After serum treatment, cells were incubated for 3 h with pHrodo redlabeled ACs (2 × 106/ml). Cells were stained and analyzed by flow cytometry. (B) Representative histograms of pHrodo expression to measure phagocytosis. Top panel, CD11c1BDCA11 DCs; bottom panel, CD141 monocytes. Blue line, untreated/full serum; red line, Ig-depleted serum. (C and D) Quantification of phagocytosed ACs measured as percentage of CD11c1BDCA11pHrodo1 cells (C) or CD141 pHrodo1 monocytes. Data represent the mean (± SEM) of two independent experiments, n 5 13 (SLE) and n 5 8 (healthy), by two-way ANOVA. (E and F) Increased BDCA11SCARF11 cells after Ig depletion. Total number of CD11c1BDCA11SCARF11 (E) or CD141 monocytes (F) mea- sured by flow cytometry in the presence of full serum or Ig-depleted serum. Data represent the mean (± SEM) of two independent experiments, n 5 13 (SLE) and n 5 8 (healthy), by two-way ANOVA.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Binding Assay, Western Blot, Dot Blot, Recombinant, Membrane, Control, Incubation, Staining, Flow Cytometry, Expressing

Journal: Cell Death & Disease

Article Title: Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c + dendritic cells

doi: 10.1038/s41419-022-05191-z

Figure Lengend Snippet: Representative serial section immunohistochemical (IHC) staining of control/non-fibrotic (left panel) and fibrotic (middle panel) kidney tissue probed for ( A ) aquaporin-1, ( B ) GPX4, ( C ) 4-HNE and ( D ) CD1c. Regions of CD1c + DC co-localisation with aqupaporin-1 + PTEC displaying evidence of ferroptotic cell death (↓ GPX4 and ↑ 4-HNE) are highlighted with black arrows. Scale bars represent 60 µm. Quantitative analysis (positive pixel intensity/µm 2 total area) of IHC staining in control and fibrotic tissue is presented (right panels). Symbols represent values for individual donor tissue. Results represent mean ± SEM of values from four randomly selected areas for each tissue sample. * P < 0.05, *** P < 0.001, Welch’s t test.

Article Snippet: Serial sections were probed with antibodies against AQP-1 (Cat. No. sc-20810; Santa Cruz Biotechnology, Dallas, TX, USA), GPX4 (Cat. No. ab41787; Abcam, Cambridge, MA, USA), 4-HNE (Cat. No. ab46544; Abcam) or CD1c (Cat. No. AF5910; R&D Systems, Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Immunohistochemistry, Control

Journal: Cell Death & Disease

Article Title: Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c + dendritic cells

doi: 10.1038/s41419-022-05191-z

Figure Lengend Snippet: A Heatmap representing secreted cytokine levels following 24-h co-culture (DC & PTEC) of flow cytometry sorted CD1c + DC with pre-conditioned normoxic (N; 21% O 2 ) or hypoxic (H; 1% O 2 ) PTEC in the absence (Nil) or presence of poly I:C. PTEC alone and DC alone cultures are included as controls; n = 4. The colour bar represents the z-score. Yellow indicates low expression; purple indicates high expression. IL-12p70, IL-17, interferon (IFN)-α and IFN-γ were not detectable in any culture conditions (data not shown). B , C Secreted IL-1β ( B ) and IL-18 ( C ) protein levels (measured by LEGENDplex™ assay; pg/ml) following 24-h co-culture (DC & PTEC) of flow cytometry sorted CD1c + DC with pre-conditioned normoxic (white bars) or hypoxic (grey bars) PTEC in the absence (Nil) or presence of poly I:C. PTEC alone and DC alone cultures are included as controls; N.D. not detectable. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 4. **** P < 0.0001, one-way ANOVA with Tukey’s multiple-comparison test. D Flow cytometric detection of intracellular IL-1β protein following 4-h co-culture of magnetic bead-enriched CD1c + DC with pre-conditioned hypoxic PTEC in the presence of poly I:C. Co-cultures were treated prior to the 4-h culture period with either brefeldin A or VX-765 to enable intracellular IL-1β accumulation/detection. Representative flow cytometric histograms of IL-1β staining (black unfilled) compared with unstained control (grey filled) for PTEC (gated on live, single, CD45 − cells) and CD1c + DC (gated on live, single, CD45 + lineage − HLA-DR + CD1c + cells) are presented. One representative of three individual donor PTEC experiments is shown. E Fold changes (relative to –Fer-1) in secreted IL-1β levels (measured by ELISA; pg/ml) following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c + DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (-Fer-1) or presence of ferrostatin-1 (+Fer-1). Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 7. ns not significant, paired t test.

Article Snippet: Serial sections were probed with antibodies against AQP-1 (Cat. No. sc-20810; Santa Cruz Biotechnology, Dallas, TX, USA), GPX4 (Cat. No. ab41787; Abcam, Cambridge, MA, USA), 4-HNE (Cat. No. ab46544; Abcam) or CD1c (Cat. No. AF5910; R&D Systems, Minneapolis, MN, USA).

Techniques: Co-Culture Assay, Flow Cytometry, Expressing, Comparison, Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c + dendritic cells

doi: 10.1038/s41419-022-05191-z

Figure Lengend Snippet: A NLRP3 mRNA expression relative to housekeeping gene β-2-microglobulin ( B2M) in flow cytometry sorted CD1c + DC freshly isolated (0 h) or following 24-h co-culture (DC & PTEC) with pre-conditioned normoxic (white bar) or hypoxic (grey bar) PTEC in the presence of poly I:C. DC alone cultures are included as controls. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 7. * P < 0.05, one-way ANOVA with Tukey’s multiple-comparison test. B Heatmap representing secreted cytokine levels following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c + DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (NIL) or presence of inflammasome inhibitors (VX-765, MCC950) or DAMP inhibitors (apyrase, isotype control or HMGB1 antibody); n = 6. The colour bar represents the z-score. Yellow indicates low expression; purple indicates high expression. C , D Fold changes (relative to NIL inhibitor) in secreted IL-1β ( C ) and IL-18 ( D ) protein levels (measured by LEGENDplex™ assay) following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c + DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (NIL) or presence of inflammasome inhibitors (VX-765, MCC950) or DAMP inhibitors (apyrase, isotype control or HMGB1 antibody). Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 6. * P < 0.05; ** P < 0.01; **** P < 0.0001, one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: Serial sections were probed with antibodies against AQP-1 (Cat. No. sc-20810; Santa Cruz Biotechnology, Dallas, TX, USA), GPX4 (Cat. No. ab41787; Abcam, Cambridge, MA, USA), 4-HNE (Cat. No. ab46544; Abcam) or CD1c (Cat. No. AF5910; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Flow Cytometry, Isolation, Co-Culture Assay, Comparison, Control

Journal: Cell Death & Disease

Article Title: Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c + dendritic cells

doi: 10.1038/s41419-022-05191-z

Figure Lengend Snippet: A , B Immunofluorescent labelling of frozen sections from control/non-fibrotic ( A ) and fibrotic kidney tissue ( B ) stained for ASC (green), CD1c (red), aquaporin-1 (AQP-1) (white) and DAPI (blue). Scale bars represent 50 µm for small frames (left panels) and 20 µm for large frames (right panels). ASC-speck-positive CD1c + DC are highlighted with white arrows. C Quantification (mean cells/mm 2 ) of ASC-speck-negative CD1c + DC (white bars) and ASC-speck-positive CD1c + DC (grey bars) in control/non-fibrotic kidney tissue ( n = 4) and fibrotic kidney tissue ( n = 4). Symbols represent values for individual donor tissue. Results represent mean ± SEM of values from five randomly selected areas for each tissue sample. * P < 0.05, ** P < 0.01, Welch’s t test.

Article Snippet: Serial sections were probed with antibodies against AQP-1 (Cat. No. sc-20810; Santa Cruz Biotechnology, Dallas, TX, USA), GPX4 (Cat. No. ab41787; Abcam, Cambridge, MA, USA), 4-HNE (Cat. No. ab46544; Abcam) or CD1c (Cat. No. AF5910; R&D Systems, Minneapolis, MN, USA).

Techniques: Control, Staining

Journal: The Journal of Investigative Dermatology

Article Title: Phenotypic Characterization of Leukocytes in Prenatal Human Dermis

doi: 10.1038/jid.2012.187

Figure Lengend Snippet: CD206 + CD1c + DDCs can be distinguished from CD206 + CD209 + skin macrophages in embryonic skin. ( a ) Arrows indicate CD206 + CD209 + cells, and arrowheads indicate CD1c + DCs in all indicated age groups. One of at least four experiments per group is shown. Bars=50 μm. ( b ) Dot graph shows the increase of CD45 + CD1c + cells in developing skin using flow cytometry. ( c ) Shown are relative numbers of CD1c + and CD209 + dermal cells at selected developmental time points analyzed by immunoflourescence ( n =4–5 per group). DC, dendritic cell; DDC, dermal dendritic cell; EGA, estimated gestational age.

Article Snippet: Fixed sections were stained with uncongujated primary antibodies anti-CD1c (L161; Serotec, Kidlington, UK), anti-CD209 (DCN46, Becton Dickinson), or FoxP3 (236A/E7; eBioscience, San Diego, CA) overnight at 4 °C and then detected with donkey-anti-mouse NorthernLight637 (R&D Systems).

Techniques: Flow Cytometry

Journal: The Journal of Investigative Dermatology

Article Title: Phenotypic Characterization of Leukocytes in Prenatal Human Dermis

doi: 10.1038/jid.2012.187

Figure Lengend Snippet: CD11c + leukocytes are present throughout skin development. ( a , b ) Multiparameter flow cytometry of freshly isolated single cell suspensions of embryonic, fetal, and adult skin was performed by incubation with mAbs against the cell surface markers indicated. Gates in dot plots were set according to isotype-matched control staining. Dead cells were excluded by 7-amino-actinomycin-D uptake. Dot plots are representative of 3–6 experiments. ( b ) CD45 + HLA-DR high (R1), CD45 + HLA-DR weak (R2), and CD45 + HLA-DR − (R3) cells from the indicated age groups are compared with regard to expression of CD11c and CD1c (lower panels). EGA, estimated gestational age.

Article Snippet: Fixed sections were stained with uncongujated primary antibodies anti-CD1c (L161; Serotec, Kidlington, UK), anti-CD209 (DCN46, Becton Dickinson), or FoxP3 (236A/E7; eBioscience, San Diego, CA) overnight at 4 °C and then detected with donkey-anti-mouse NorthernLight637 (R&D Systems).

Techniques: Flow Cytometry, Isolation, Incubation, Control, Staining, Expressing