cd166 fc  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Elevated hepatic and serum ALCAM were observed in patients with AIH. (A) Representative immunohistochemistry images of ALCAM in liver biopsies from HC (n=3) and patients with AIH (n=6). (B) Representative immunofluorescence staining for CD4, CD6 and ALCAM in interface hepatitis lesion of liver sections from patients with AIH (n=3). (C) Concentration of serum ALCAM in HC (n=28) and AIH (n=86) was measured by ELISA assay. Individual correlation between clinical indicators and serum ALCAM was calculated in patients with AIH (n=86). (D) Correlation between the number of hepatic CD6 + cells and paired serum ALCAM concentration was calculated (n=27). ***p < 0.001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: ALCAM promoted CD6 high CD4 + T cells trans-endothelial migration in vitro . (A, B) Human CD4 + T cells were magnetically isolated from PBMC of healthy donors and stimulated with αCD3/28 for 3 days in a flat-bottom 96-well plate, the expression of CD6 and cell proliferation was detected with flow cytometry. (C) After stimulation, the expression of cytokines, surface markers and transcription factors was compared between the CD6 high and CD6 low subsets. (D) Pre-activated CD4 + T cells were placed on a transwell chamber with 5μm pore for 24 hours in the presence of rhALCAM (3ug/ml) or vehicle (PBS). The expression of CD6 and CD69 was measured by flow cytometry. Experiments were repeated at least three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration, In Vitro, Isolation, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Schematic diagram for this study. Upregulated ALCAM on hepatocytes promoted the trans-endothelial migration of pathogenic CD6 high CD4 + T cells, which further aggravated hepatic inflammation of patients with AIH. This study revealed a putative therapeutic approach for patients with AIH.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration

Journal: Stem cells (Dayton, Ohio)

Article Title: CD166 engagement augments mouse and human hematopoietic progenitor function via activation of stemness and cell cycle pathways

doi: 10.1002/stem.3053

Figure Lengend Snippet: (A) Illustration of OB, CD166+ and CD166− LSK cell isolation, co-cultures and analysis. Preparation of OBs was initiated 7 days before seeding at time 0 (D0) with 1000 freshly sorted C57BL/6–derived CD166+ or CD166− LSK cells per well. OBs were cultured in a-MEM medium with 10% FBS, 1% Penicillin-Streptomycin, 1% L-glutamine. Once CD166+ and CD166− LSK cells were seeded, the medium was changed to a 1:1 mixture of OB culture medium (a-MEM as described above) and LSK cell culture medium (IMDM medium with 10% FBS, 10 ng/mL recombinant murine SCF, 10 ng/mL IL-3, 20 ng/mL insulin-like growth factor 1 (IGF-1), 20 ng/mL TPO, 25 ng/mL IL-6 and 25 ng/mL Flt3L). CFU-GM (B), BFU-E (C), CFU-GEMM (D), total CFU (E), and CFU fold change (F) between D0 numbers and those obtained on D7 from the progeny of 1000 LSK cells. CFU fold increase was calculated relative to that obtained from 1000 freshly isolated CD166+ and CD166− LSK cells plated on D0. Data are pooled from 2 independent experiments, each performed in triplicates. *p < 0.05, **p < 0.01.

Article Snippet: Recombinant mouse or human CD166 protein treatment For CFU and phenotyping assays, 48-well culture plates were coated with 10 μg/mL recombinant mouse (rmCD166; R&D, 1172-AL-050) or human CD166 protein (rhCD166; R&D, 656-AL-100) in Eagle’s balanced salt solution overnight at 4°C.

Techniques: Cell Isolation, Derivative Assay, Cell Culture, Recombinant, Isolation

Journal: Stem cells (Dayton, Ohio)

Article Title: CD166 engagement augments mouse and human hematopoietic progenitor function via activation of stemness and cell cycle pathways

doi: 10.1002/stem.3053

Figure Lengend Snippet: (A) Schematic diagram of rmCD166 coating, CD166+ and CD166− LSK cell isolation, culture and analysis. Coating of rmCD166 (or BSA as control) was initiated 1 day before seeding at time 0 (D0) with 500 freshly sorted C57BL/6–derived CD166+ or CD166− LSK cells per well. CD166+ and CD166− LSK cells were cultured in IMDM medium with 10% FBS. Medium was supplemented with exogenous cytokines as described in the legend of Figure 1. CFU-GM (B), BFU-E (C), CFU-GEMM (D), total CFU (E), and CFU fold change (F) between D0 numbers and those obtained on D7 from the progeny of 500 LSK cells. CFU fold increase was calculated relative to that obtained from 500 freshly isolated CD166+ LSK and CD166− LSK cells assayed on D0. (G) Flow cytometric analyses of the Lin− Sca-1+ population and comparison of absolute numbers of Lin− Sca-1+ cells on D7 of culture. Data pooled from 4 independent experiments, each performed in triplicates. *p < 0.05, **p < 0.01. Please note that all cultures were supplemented with exogenous cytokines, and therefore it is expected to observe a CFU-increase regardless of what substrate is used.

Article Snippet: Recombinant mouse or human CD166 protein treatment For CFU and phenotyping assays, 48-well culture plates were coated with 10 μg/mL recombinant mouse (rmCD166; R&D, 1172-AL-050) or human CD166 protein (rhCD166; R&D, 656-AL-100) in Eagle’s balanced salt solution overnight at 4°C.

Techniques: Cell Isolation, Control, Derivative Assay, Cell Culture, Isolation, Comparison

Journal: Stem cells (Dayton, Ohio)

Article Title: CD166 engagement augments mouse and human hematopoietic progenitor function via activation of stemness and cell cycle pathways

doi: 10.1002/stem.3053

Figure Lengend Snippet: (A) Schematic representation of rhCD166 protein treatment, CD34+, CD34+CD166+, CD34+CD166− cell isolation, culture and analysis. Coating of 48-well culture plates with 10 μg/mL rhCD166 (BSA as control) in Eagle’s balanced salt solution overnight at 4 °C was initiated 1 day before seeding at time 0 (D0) with 4000 freshly sorted CD34+, CD34+CD166+, CD34+CD166− cells per well. Cultures were cultured in StemSpan™ SFEM II supplemented with human cytokines: 50 ng/ml TPO, 100 ng/ml SCF, and Flt3L and incubated at 5% O2, 5% CO2. Analysis of CFU-GM (B), CFU-GEMM (C), total CFU (D), and CFU fold change (E) between D0 numbers and those obtained on D7 from the progeny of 4000 cells from each phenotype. CFU fold increase was calculated relative to that obtained from 4000 freshly isolated CD34+, CD34+CD166+, and CD34+CD166− cells plated on D0. Data is pooled from at least 4 independent experiments, each performed in triplicates. *p < 0.05, **p < 0.01.

Article Snippet: Recombinant mouse or human CD166 protein treatment For CFU and phenotyping assays, 48-well culture plates were coated with 10 μg/mL recombinant mouse (rmCD166; R&D, 1172-AL-050) or human CD166 protein (rhCD166; R&D, 656-AL-100) in Eagle’s balanced salt solution overnight at 4°C.

Techniques: Cell Isolation, Control, Cell Culture, Incubation, Isolation

Journal: Stem cells (Dayton, Ohio)

Article Title: CD166 engagement augments mouse and human hematopoietic progenitor function via activation of stemness and cell cycle pathways

doi: 10.1002/stem.3053

Figure Lengend Snippet: (A) Schematic illustration of rmCD166 protein treatment, wide type (WT) and CD166 knockout (CD166−/−) LSK cell isolation, culture and analysis. Coating of 48-well culture plates with 10 μg/mL rmCD166 (BSA as control) in Eagle’s balanced salt solution overnight at 4 °C was initiated 1 day before seeding at time 0 (D0) with 500 freshly sorted WT LSK and CD166−/− LSK cells per well. Cultures were incubated at 5% O2, 5% CO2 and supplemented with exogenous cytokines as detailed in Figure 1. CFU-GM (B), BFU-E (C) CFU-GEMM (D), total CFU (E), and CFU fold change (F) between D0 numbers and those obtained on D7 from the progeny of 500 LSK cells. CFU fold increase was calculated relative to that obtained from 500 freshly isolated WT LSK and CD166−/− LSK cells assayed on D0. Data are pooled from 3 independent experiments, each performed in triplicates. *p < 0.05, **p < 0.01.

Article Snippet: Recombinant mouse or human CD166 protein treatment For CFU and phenotyping assays, 48-well culture plates were coated with 10 μg/mL recombinant mouse (rmCD166; R&D, 1172-AL-050) or human CD166 protein (rhCD166; R&D, 656-AL-100) in Eagle’s balanced salt solution overnight at 4°C.

Techniques: Knock-Out, Cell Isolation, Control, Incubation, Isolation

Journal: Stem cells (Dayton, Ohio)

Article Title: CD166 engagement augments mouse and human hematopoietic progenitor function via activation of stemness and cell cycle pathways

doi: 10.1002/stem.3053

Figure Lengend Snippet: (A) Analysis of the 3 data sets from 3 separate cell types as indicated in the Figure legend on a tSNE plot. (B) PCA plot of significantly expressed genes after cell cycle correction. (C and D) PCA and tSNE plots of the differentially expressed genes suggest distinct differences between CD166−/− and WT cells. (E and F) Predicted cell trajectories using genes of mitochondria-mediated signaling, Epigenetic, Cytokine,growth factor and hormone signaling, Cell communication, Cell cycle signaling, Metabolic, Translational regulation, and Stem Cell Pluripotency. (G) Gene expression profile of selected genes. (H, I) Gene expression profile of differentially expressed stem cell pluripotency and metabolic genes. CD166 KO and WT samples are colored by green and pink, respectively, on the column side color bar.

Article Snippet: Recombinant mouse or human CD166 protein treatment For CFU and phenotyping assays, 48-well culture plates were coated with 10 μg/mL recombinant mouse (rmCD166; R&D, 1172-AL-050) or human CD166 protein (rhCD166; R&D, 656-AL-100) in Eagle’s balanced salt solution overnight at 4°C.

Techniques: Gene Expression

Journal: Stem cells (Dayton, Ohio)

Article Title: CD166 engagement augments mouse and human hematopoietic progenitor function via activation of stemness and cell cycle pathways

doi: 10.1002/stem.3053

Figure Lengend Snippet: Data from two independent experiments showing chimerism levels at 8 weeks and 16 weeks post-transplantation. Each symbol represents a single mouse. CD166+ LSK cells were cultured for 5 days on plates covered with immobilized CD166 protein or BSA then co-transplanted with 250,000 BoyJ (CD45.1) BM mononuclear competitor cells. n=17 for rmCD166 and n=18 for BSA groups.

Article Snippet: Recombinant mouse or human CD166 protein treatment For CFU and phenotyping assays, 48-well culture plates were coated with 10 μg/mL recombinant mouse (rmCD166; R&D, 1172-AL-050) or human CD166 protein (rhCD166; R&D, 656-AL-100) in Eagle’s balanced salt solution overnight at 4°C.

Techniques: Transplantation Assay, Cell Culture