Journal: Cell reports

Article Title: PAI-1 uncouples integrin-β1 from restrain by membrane-bound β-catenin to promote collagen fibril remodeling in obesity-related neoplasms

doi: 10.1016/j.celrep.2024.114527

Figure Lengend Snippet:

Article Snippet: For DKK1-, SPARC-, TIMP2-, COL1, integrin β2, or integrin α3 PLA, purified rabbit monoclonal anti-DKK1 (1:200, Cell Signaling Technology, Cat# 48367S), rabbit polyclonal anti-SPARC (1:200, Cell Signaling Technology, Cat# 5420S), mouse monoclonal anti-TIMP2 (1:200, R&D Systems, Cat# MAB971), mouse monoclonal anti-COL1 (1:200, Cell Signaling Technology, Cat# 66948S), goat polyclonal anti-integrin β1 (1:200, R&D Systems, Cat# AF1778), goat polyclonal anti-integrin β2 (1:200, R&D Systems, Cat# AF1730), and mouse monoclonal anti-integrin α3 (1:200, R&D Systems, Cat# MAB1345) were used.

Techniques: Recombinant, Membrane, Cell Culture, Reverse Transcription, SYBR Green Assay, Clinical Proteomics, Protein Extraction, Extraction, Bicinchoninic Acid Protein Assay, In Situ, Blocking Assay, Migration, shRNA, Control, Software, Pyromark Assay, Western Blot, Simple Western

Journal: Frontiers in Veterinary Science

Article Title: Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs

doi: 10.3389/fvets.2024.1405297

Figure Lengend Snippet: Conjugated antibodies used for triple-labeling cells in acute leukemia immunophenotyping panels after April 2021, including their target antigen and registry number (when available) or source.

Article Snippet: CD11b-FITC/CD34-PE/CD18-A647* , CD18 : Bio-Rad Cat# MCA503A647, RRID:AB_324799.

Techniques:

Journal: Fundamental Research

Article Title: A dynamic peripheral immune landscape during human pregnancy

doi: 10.1016/j.fmre.2022.06.011

Figure Lengend Snippet: Aberrant cell-cell communication supported the decreased adaptive immune response after pregnancy. (a) Dot plot showing the representative GO biological process and pathways enriched in downregulated DEGs in DC and MC subsets. (b) Venn plot showing the integrated comparative analysis of downregulated DEGs among DC and MC subsets. (c) Circle plot showing the inferred MHC-II signaling networks. (d) Violin plot showing the expression of genes related to MHC-II signaling among immune populations between HC (blue) and Preg (red) groups. (e) Circle plot showing the inferred CD40 signaling networks. (f) Circle plot showing the inferred ICOS signaling networks in Preg group but absent in HC group. (g) Violin plot showing the expression of genes related to ICOS signaling among immune populations between HC (blue) and Preg (red) groups. (h) Circle plot showing the inferred MIF signaling networks. (i) Violin plot showing the expression of genes related to MIF signaling among immune populations between HC (blue) and Preg (red) groups. (j) The flow cytometry histogram showing the expression of CD74 in CD4 + TCs. Box plots showing the ratio of CD74-expressing CD4 + TCs between HC and Preg groups. (k) Violin plot showing the expression of genes related to ICAM signaling among immune populations between HC (blue) and Preg (red) groups. (l) The flow cytometry histogram showing the expression of CD18 in CD4 + TCs (left). Box plots showing the Mean Fluorescence Intensity (MFI) of CD18 in CD4 + TCs between HC and Preg groups (right). Significance in j, l was calculated using an unpaired, two-tailed Student's t-test; * P < 0.05.

Article Snippet: For the surface staining, PBMCs were simultaneously stained with anti-human fluorochrome-labeled antibodies specific for CD45 (BV785, #304048), CD3 (BV421, #300434), CD14 (PerCP/Cy5.5, #301824), CD4 (APC, #317416 or PE, #300508), CD8 (PerCP/Cy5.5, #301031), CD45RA (BV785, #304140), CD45RO (BV605, #304238), CD1C (PerCP/Cy5.5, #331514), CD19 (BV605, #302244), CD20 (APC, #302310), CD38 (BV785, #303530), HLA-DQ (PE, #318105), BCMA (PE-Cy7, #357508), CD74 (APC, #326811), CD69 (BV650, #310934), CD83 (PE, #305307) (BioLegend, San Diego, USA) and CD18 (APC, #E-AB-F1057E) (Elabscience).

Techniques: Expressing, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Primer sequences and PCR settings

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Hybridization

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Expression of CD11a and CD18 in melanoma A375, 1205LU and SLM8 cell lines with conditioned medium. Cell-surface expression of CD11a and CD18 on indicated melanoma cell lines treated for 24 hrs with HUVEC conditioned medium was analyzed by flow cytometry. Isotypic controls are represented as empty histograms and specific antibody-labelling is displayed as shaded histograms. Histograms obtained with cells incubated with FCS-complete medium and labeled with specific antibodies, which overlap with the isotypic control are not shown. Data from obtained with 3 independent experiments.

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Expressing, Flow Cytometry, Incubation, Labeling, Control

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Effect of CD11a and CD18-blocking antibodies on the transendothelial migration of A375, 1205LU and SLM8 cell lines. The experiments were performed as detailed in Figure , except that 2μg/ml of CD11a or CD18-blocking antibodies were introduced in the upper chamber of the Transwells when indicated. Histograms represent 3 independent experiments. In each experiment each condition was analyzed in duplicate.

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Blocking Assay, Migration

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Effect of CD11 and CD18-blocking antibodies on the formation of clumps. A Semi-quantitative PCRs were performed to detect the expression of the ICAM-1 transcript. GAPDH is used as a DNA amount control. B A375, 1205LU and SLM8 cell lines were treated with 2 μg/ml of CD11a or CD18-blocking antibodies as indicated. Melanoma cells were labeled with DiO then fixed and labeled with DAPI prior to their observation under an epifluorescence microscope using a magnification of x10. Data were obtained from 3 independent experiments.

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Blocking Assay, Expressing, Control, Labeling, Microscopy

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Histology of the vastus lateralis muscle from a 6-month-old DE50-MD dog. (A-E) Immunohistochemistry of a section of vastus lateralis muscle showing (A) CCL2 (magenta), (B) CD18 (green) and (C) a merged image of CCL2, CD18 and Hoechst 33342 (blue), with magnified views of the boxed region in C shown for (D) CCL2 and (E) CD18 staining. (F-J) Immunohistochemistry of a serial section showing (F) CCL2 (magenta), (G) MAC387 + regions (green) and (H) a merged image of CCL2, MAC387 and Hoechst 33342 (blue), with magnified views of the boxed region in H shown for (I) CCL2 and (J) CD18 staining. (K) A serial section with H&E staining showing cell infiltrate within and between muscle fibres, fibres with internalised nuclei and hypercontracted fibres (darker magenta). (L) Serial section with acid phosphatase (AP) staining showing positive red staining at areas of cell infiltrate and within some muscle fibres. (M-O) Serial section of immunohistochemistry with no primary antibody as a control with magnified views of the region of inflammation (dashed box) shown for the (N) magenta (CCL2) and (O) green (CD18/MAC387) channels. Scale bar: 100 µm.

Article Snippet: Sections were then incubated with the following combinations of primary antibodies for 1 h at room temperature: goat anti-canine CCL2 IgG (R&D systems, AF1774, 1:20) with rat anti-human perlecan (Millipore, clone A7L6, 1:1000) , mouse anti-canine CD18 (Bio-Rad, monoclonal IgG1, clone CA1.4E9, MA1-82363, 1:100) ( C) or mouse anti-human macrophages (Biorad, monoclonal IgG1, clone MAC387, MCA874GT, 1:100) ( D).

Techniques: Immunohistochemistry, Staining, Control

Journal: Journal of Leukocyte Biology

Article Title: Mitofusin-2 regulates leukocyte adhesion and β2 integrin activation

doi: 10.1002/jlb.1a0720-471r

Figure Lengend Snippet: FIGURE 2 Mitofusin-2 knockdown reduces fMLP receptor expression as well as receptor-dependent and independent β2 integrin activation in neutrophil-like HL60 cells. (A) Mean ± SD of surface FPR1 expression on Control or MFN2-knockdown (MFN2 KD) HL60 cells from n = 3 individual experiments. (B-I) Mean ± SD of overall surface CD18 (β2 integrins, B,C), CD11a (αL integrins, D,E), CD11b (αM integrins, F,G), CD11c (αX integrins, H,I) expression on Control or MFN2 KD HL60 cells stimulated with FPR1-dependent fMLP (100 nM, at RT for 20 min, closed bars, B, D, F, H), receptor-independent PMA (100 nM, at RT for 20 min, gray bars, C, E, G, I), or vehicle control (open bars) from n = 3 individual experiments. (J-U) Mean ± SD of high-affinity (H+, mAb24 staining, J-M) and extended (E+, KIM127 staining, N-Q) β2 integrin as well as H+ αM integrin (CBRM1/5 staining, R-U) expression on Control or MFN2 KD HL60 cells stimulated with FPR1-dependent fMLP (100 nM, at RT for 20 min, closed bars, J, L, N, P, R, T), receptor-independent PMA (100 nM, at RT for 20 min, gray bars, K, M, O, Q, S, U) or vehicle control (open bars) from n = 3 individual experiments. In L,M; P,Q; and T,U, the MFI of mAb24, KIM127, and CBRM1/5 is normalized to overall expression of CD18 (B,C), CD18 (B,C), and CD11b (F,G), respectively. n.s., non-significant (P > 0.05). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t-test (A) or 2-way ANOVA followed by Tukey’s multiple comparisons test (B-U). HL60 cells were pre-differentiated with 1.3% DMSO for 7 days

Article Snippet: Recombinant human P-selectin-Fc, ICAM-1-Fc, recombinant mouse ICAM-1-Fc, and Alexa Fluor 647 (AF647)-conjugated mouse antihuman CD18 (pan-β2 integrins) antibody (Ab) were purchased from R&D Systems.

Techniques: Knockdown, Expressing, Activation Assay, Control, Staining

Journal: Journal of Leukocyte Biology

Article Title: Mitofusin-2 regulates leukocyte adhesion and β2 integrin activation

doi: 10.1002/jlb.1a0720-471r

Figure Lengend Snippet: FIGURE 4 Mitofusin-2 knockdown inhibited Mn2+-induced integrin-outside-in-signaling-dependent spreading of neutrophil-like HL60 cells. (A) Representative total internal reflection fluorescence images of spreading in Control or MFN2-knockdown (MFN2 KD) HL60 cell footprints pretreated with anti-CD18 blocking antibody or isotype control. Cells were stained with CellTracker Orange CMRA. Spreading was induced by 1 μM Mn2+ on coated ICAM-1. The scale bar is 10 μm. (B) Mean ± SD of the cell footprint area of n = 122 (control, isotype), n = 114 (control, CD18 Ab), n = 118 (MFN2 KD, isotype), and n = 134 (MFN2 KD, CD18 Ab) cells, respectively, from 6 independent records per group. n.s., non-significant (P > 0.05) ***P < 0.001, ****P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple comparisons test. HL60 cells were pre-differentiated with 1.3% DMSO for 7 days

Article Snippet: Recombinant human P-selectin-Fc, ICAM-1-Fc, recombinant mouse ICAM-1-Fc, and Alexa Fluor 647 (AF647)-conjugated mouse antihuman CD18 (pan-β2 integrins) antibody (Ab) were purchased from R&D Systems.

Techniques: Knockdown, Fluorescence, Control, Blocking Assay, Staining

Journal: Journal of Leukocyte Biology

Article Title: Mitofusin-2 regulates leukocyte adhesion and β2 integrin activation

doi: 10.1002/jlb.1a0720-471r

Figure Lengend Snippet: FIGURE 5 The maturation of β2 integrin activation during the DMSO-induced differentiation of HL60 cells. (A) Contour plots showing expression of high-affinity (H+, mAb24 staining) and extended (E+, KIM127 staining) β2 integrins on HL60 cells (upper panels) or primary human neutrophils (lower panel) stimulated with fMLP (1 mM, right panels) or vehicle control (left panels). HL60 cells were pre-differentiated with 1.3% DMSO for indicated days. (B,C) Expression of mAb24 (B) and KIM127 (C) on HL-60 cells after different days of DSMO-induced differentiation with or without fMLP stimulation. (D) The percentage of mAb24+KIM127+ HL-60 cells after different days of DSMO-induced differentiation with or without fMLP stimulation. (E) Expression of overall CD18 (conformation unspecific) on HL-60 cells after different days of DSMO-induced differentiation with or without fMLP stimulation. (F) FPR1 expression on HL-60 cells after different days of DSMO-induced differentiation. Mean ± 95%CI for n = 3 individual experiments in (B-G), **P < 0.01, ***P < 0.001, ****P < 0.0001 by comparing fMLP stimulated cells and vehicle control using 2-way ANOVA followed by Sidak’s multiple comparisons test

Article Snippet: Recombinant human P-selectin-Fc, ICAM-1-Fc, recombinant mouse ICAM-1-Fc, and Alexa Fluor 647 (AF647)-conjugated mouse antihuman CD18 (pan-β2 integrins) antibody (Ab) were purchased from R&D Systems.

Techniques: Activation Assay, Expressing, Staining, Control

Journal: Journal of Leukocyte Biology

Article Title: Mitofusin-2 regulates leukocyte adhesion and β2 integrin activation

doi: 10.1002/jlb.1a0720-471r

Figure Lengend Snippet: FIGURE 6 Mitofusin-2 knockdown impairs expression of β2-integrin-activation maturation markers during the DMSO-induced differentiation of HL60 cells. (A,B) Representative histograms showing expression of CD35 (A) and CD87 (B) on HL60 cells pre-differentiated for 5 days (dark gray) or not (light gray). (C-D) Expression of CD35 (C), and CD87 (D) on HL-60 cells after different days of DSMO-induced differentiation. Mean ± 95%CI for n = 3 individual experiments. (E-H) Contour plots showing expression of high-affinity (H+, mAb24 staining) β2 integrins on pre-differentiated HL60 cells with different expression of CD35 (E), CD87 (F), and overall CD18 (G) stimulated with fMLP (100 nM, right panels) or vehicle control (left panels). (H) The percentage of mAb24+ cells in the low-expression or high-expression population of different markers upon fMLP stimulation. (I-K) Expression of CD35 (I) and CD87 (J-K) on Control or MFN2-knockdown (MFN2 KD) HL60 cells after pre-differentiated for 5 (I-J) or 7 (K) days. Mean ± SD for n = 3 (I) or 9 (J-K) individual experiments, *P < 0.05, ****P < 0.0001 by Student’s t-test

Article Snippet: Recombinant human P-selectin-Fc, ICAM-1-Fc, recombinant mouse ICAM-1-Fc, and Alexa Fluor 647 (AF647)-conjugated mouse antihuman CD18 (pan-β2 integrins) antibody (Ab) were purchased from R&D Systems.

Techniques: Knockdown, Expressing, Activation Assay, Staining, Control

Journal: Journal of Leukocyte Biology

Article Title: Mitofusin-2 regulates leukocyte adhesion and β2 integrin activation

doi: 10.1002/jlb.1a0720-471r

Figure Lengend Snippet: FIGURE 8 Mitofusin-2 knockdown inhibits β2 integrin activation and adhesion of HL60 cells at the same maturation level. (A) Mean ± SD of surface CD87 expression on CD87high Control and MFN2-knockdown (MFN2 KD) HL60 cells from n = 6 individual experiments. (B) Mean ± SD of nucleus segment number of CD87high Control (n = 21) and MFN2 KD (n = 19) HL60 cells from 9 and 11 independent records, respectively. (C and D) Mean ± SD of MFN2 and FPR1 expression on CD87high Control and MFN2 KD HL60 cells from n = 3 individual experiments. (E and F) Mean ± SD of overall surface CD18 (β2 integrins) expression on CD87high control or MFN2 KD HL60 cells stimulated with FPR1-dependent fMLP (100 nM, at RT for 20 min, closed bars, E), receptor-independent PMA (100 nM, at RT for 20 min, gray bars, F), or vehicle control (open bars) from n = 3 individual experiments. (G-N) Mean±SD of high-affinity (H+, mAb24 staining, G-J) and extended (E+, KIM127 staining, K-N) β2 integrin expression on CD87high control or MFN2 KD HL60 cells stimulated with FPR1-dependent fMLP (100 nM, at RT for 20 min, closed bars, G, I, K, M), receptor-independent PMA (100 nM, at RT for 20 min, gray bars, H, J, L, N), or vehicle control (open bars) from n = 3 individual experiments. In (I and J) and (M and N), the MFI of mAb24 and KIM127 is normalized to overall expression of CD18 (E and F). (O and P) A cumulative histogram (O) and a bar graph (P, mean ± SD, n = 46 cells from 3 individual experiments per group) showing the rolling velocity of CD87high Control or MFN2 KD HL60 cells rolling on the substrate of P-selectin+ICAM-1 under a wall shear stress of 6 dyn/cm2. (Q) The number of arrested CD87high Control or MFN2 KD HL60 cells on the substrate of P-selectin/ICAM-1 with or without the stimulation of fMLP (100 nM) under a wall shear stress of 6 dyn/cm2. Mean ± SD, n = 17 individual records. n.s., non-significant (P > 0.05). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t-test (A-D, P) or 2-way ANOVA followed by Tukey’s multiple comparisons test (E-N, Q). HL60 cells were pre-differentiated with 1.3% DMSO for 7 days

Article Snippet: Recombinant human P-selectin-Fc, ICAM-1-Fc, recombinant mouse ICAM-1-Fc, and Alexa Fluor 647 (AF647)-conjugated mouse antihuman CD18 (pan-β2 integrins) antibody (Ab) were purchased from R&D Systems.

Techniques: Knockdown, Activation Assay, Expressing, Control, Staining, Shear