Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Cells incubated with HIV-1 particles were lysed, and cell lysates used for measuring cell-associated p24 gag . The data shown are the mean percent of captured p24 gag (virus) ± SEM of independent experiments performed in triplicates (n = 3 for DC, n = 5 for THP-1, n = 3 for Raji and HeLa). (B) CD169 expressing cell lines and mature DCs were incubated with Gag-mCherry VLPs and stained for plasma membrane bound CD169 (Surface, top panel) or total CD169 (+ Tx100, bottom panel). CD169 (green), Gag-mCherry VLP (red) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bars represent 5 μm. (C) Cells incubated with HIV-1 particles were co-cultured with CD4 + T cells to monitor HIV-1 trans-infection. Cells were lysed two days post initiation of co-culture and lysates used for measurement of luciferase activity. Values were normalized to luciferase activity observed in control cells (immature DCs or CD169 low/null control cell lines). The data shown are the means ± SEM of independent experiments performed in triplicates with CD4 + T cells from different donors (n = 3 for DC, n = 4 for THP-1, n = 3 for Raji and n = 4 for HeLa).

Article Snippet: Cells were blocked with 20% normal mouse serum, and CD169 expression was visualized with Alexa647-conjugated anti-CD169 mAb (AbD Serotec).

Techniques: Incubation, Virus, Expressing, Staining, Clinical Proteomics, Membrane, Cell Culture, Infection, Co-Culture Assay, Luciferase, Activity Assay, Control

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Sequences of wild type and mutant CD169 CTs. The asterisks represent stop codons introduced into the ORFs of the two CT mutants. (B) Western blot analysis of THP-1 cell lysates expressing either wild type or mutant CD169. (C) Cell surface expression of CD169 on THP-1 cells was measured by flow cytometry. (D) Relative cell surface expression of CD169 CT mutants was quantified and normalized to that observed with THP-1/CD169 cells. (E) Cells were challenged with HIV-1, washed and cell-associated p24 gag was measured. The data shown is the virus capture by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) normalized to that observed with THP-1/CD169 cells. (F) THP-1/CD169- or THP-1/CD169 CT mutant-mediated trans-infection was determined by measuring luciferase activity in THP—CD4 + T cell co-cultures 2 days post initiation of co-culture. The data shown is the relative virus transmission by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) to that observed with THP-1/CD169 cells. (G) Efficacy of trans-infection was calculated as trans-infection (luciferase activity) per amount of virus captured (cell-associated p24 gag ) and normalized to that observed with THP-1/CD169 cells (set as 100). The data shown are the means ± SEM of three (D to F) or four (G) independent experiments. (H) THP-1/CD169 or THP-1/CD169ΔCT4R cells were incubated with Gag-mCherry VLPs (red), washed, fixed and stained for CD169 (green) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. WT: THP-1/CD169, ΔCT: THP-1/CD169ΔCT, ΔCT4R: THP-1/CD169ΔCT4R and Vec: empty vector transduced THP-1.

Article Snippet: Cells were blocked with 20% normal mouse serum, and CD169 expression was visualized with Alexa647-conjugated anti-CD169 mAb (AbD Serotec).

Techniques: Mutagenesis, Western Blot, Expressing, Flow Cytometry, Virus, Infection, Luciferase, Activity Assay, Co-Culture Assay, Transmission Assay, Incubation, Staining, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Amino acid sequences of the CTs of wild type (WT) CD169 and mutant CD169YF are shown. Alanine to tyrosine mutation at position 1683 (in red) creates a di-aromatic motif, YF (underlined). (B) Western blot analysis for CD169 expression in THP-1/CD169 and THP-1/CD169YF cell lysates. (C) Representative FACS analysis of cell surface expression of CD169 on wild type and YF mutant expressing THP-1 cells. (D) The mean fluorescence intensity of cell surface expression of CD169 on YF mutant expressing THP-1 cells was quantified and normalized to that observed with THP-1/CD169 (wt) cells (set at 100). (E) Cells were incubated with Gag-mCherry VLPs and stained for CD81, CD63 or Lamp1 and nucleus. CD81, CD63 or Lamp1 (green), Gag-mCherry VLP (red) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. (F) Co-localization between green (CD81, CD63 or Lamp1) and red (VLPs) signals is reported as mean Pearson’s coefficient ± SEM. Each dot represents a single cell. Two-tailed P values were calculated using unpaired t-test in GraphPad Prism 5. ***: P < 0.0001. (G) Cells were challenged with HIV-1, washed and cell-associated p24 gag was measured. Virus capture observed with THP-1/CD169YF cells was normalized to that observed with THP-1/CD169 cells (WT; set as 100). (H) Cells challenged with HIV-1/Bal-luc, were washed, co-cultured with CD4 + T cells and lysed at two days post initiation of co-culture for measurement of luciferase activity. The level of virus transmission observed in THP-1/CD169 (wt)—CD4 + T cell co-cultures was set as 100. (I) Efficacy of trans-infection was calculated as trans-infection (luciferase activity) per virus capture (cell-associated p24 gag ) and is shown relative to that observed with THP-1/CD169 cells (set as 100). The data shown are the means ± SEM of four (D) or six (G to I) independent experiments.

Article Snippet: Cells were blocked with 20% normal mouse serum, and CD169 expression was visualized with Alexa647-conjugated anti-CD169 mAb (AbD Serotec).

Techniques: Mutagenesis, Western Blot, Expressing, Fluorescence, Incubation, Staining, Two Tailed Test, Virus, Cell Culture, Co-Culture Assay, Luciferase, Activity Assay, Transmission Assay, Infection

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Representative FACS analysis for CD169 expression on LPS or IFN-α-matured DCs. (B) HIV-1 capture by immature (NT), IFN-α or LPS-matured DCs was determined by measuring cell-associated p24 gag in cell lysates. (C) HIV-1 transfer to CD4 + T cells, by immature (NT), IFN-α or LPS-matured DCs was determined by measuring luciferase activity in DC—CD4 + T cell co-cultures. HIV-1 capture and transfer experiments were performed in triplicates with DCs isolated from eight independent donors. The individual dot represents a single donor and the means ± SEM are depicted. (D) LPS or IFN-α-matured DCs were incubated with fluorescent HIV-1 particles (green) and stained for CD169 (red) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. (E) Representative electron micrographs of LPS or IFN-α-matured DCs incubated with HIV-1. The bottom panels are higher magnification pictures of the area depicted within the highlighted squares in the top panels. Arrows indicate virus particles. Scale bar represents 1 μm for top panels and 500 nm for bottom panels. (F) Cells were incubated with HIV-1 and stained for HIV-1 p24 gag (green) and CD169 (red). Cells were imaged by FPALM super resolution microscopy. The top panels represent a single LPS or IFN-α matured DC while the middle panels show cross sections along the a — b line indicated in the top panels. The bottom panels are pictures enlarged from the area depicted within the highlighted (dotted) squares in the middle panels. Scale bars represent 1 μm in the top and middle panels and 500 nm in the bottom panels. LPS: LPS-treated DCs, IFN-α: IFN-α-treated DCs, Immature: immature DCs.

Article Snippet: Cells were blocked with 20% normal mouse serum, and CD169 expression was visualized with Alexa647-conjugated anti-CD169 mAb (AbD Serotec).

Techniques: Expressing, Luciferase, Activity Assay, Isolation, Incubation, Staining, Virus, Super-Resolution Microscopy

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Experimental procedure utilized for testing the susceptibility of HIV-1 particles captured by CD169 to extracellular protease treatment is depicted. (B) Representative FACS analysis of cell surface expression of CD169 on LPS-DCs. (C) Relative cell surface expression of CD169 expression was measured by flow cytometry and normalized to that observed with untreated control (NT, set at 1). The data shown are the means ± SD of five (LPS-DCs, Trp), nine (LPS-DCs, Prn) and two (IFN-DCs, Typ and Prn) independent experiments with DCs from different donors. (D) LPS or IFN-α-matured DCs, incubated with HIV-1, were treated with pronase or trypsin. The amount of virus particles left associated with cells following protease treatments was determined by measuring cell-associated p24 gag and the values were normalized to that observed with untreated cells (NT). The data shown are the means ± SD of four (LPS-DCs, Trp), seven (LPS-DCs, Prn) and two (IFN-DCs, Typ and Prn) independent experiments with DCs from different donors. (C and D) Two-tailed P values were calculated using one sample t-test in GraphPad Prism 5. *: P < 0.05, ***: P < 0.0001. Trp: trypsin-treated sample, Prn: pronase-treated sample.

Article Snippet: Cells were blocked with 20% normal mouse serum, and CD169 expression was visualized with Alexa647-conjugated anti-CD169 mAb (AbD Serotec).

Techniques: Expressing, Flow Cytometry, Control, Incubation, Virus, Two Tailed Test

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A and B) LPS or IFN-α-matured DCs incubated with fluorescent HIV-1 particles, were stained for either surface-exposed gp120 (A) or CD169 (B) on living cells (Surface, top panels) or total gp120 (A) or CD169 (B) on cells after fixation and TritonX-100 treatment (+ Tx100, bottom panels). HIV-1 particles (green), gp120 or CD169 (red) and nucleus (blue). The arrowheads indicate green HIV-1 particles in VCCs that were not stained by surface-applied antibodies. Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. (C and D) Co-localization between HIV-1 particles and gp120 (C) or CD169 (D) is reported as Manders’ coefficients. Each dot represents a single cell and the means ± SEM are shown. The data shown is a representative experiment using DCs isolated from two different donors. (E and F) HIV-1 exposed LPS or IFN-α-matured DCs or cell-free (CF) HIV-1 particles were incubated with increasing concentrations of VRC01 (E), NIH45–46 G54W (F) or sCD4–183 (G) prior to initiation of CD4 + T cell infections. The x-axis shows the concentration of input VRC01 (E), NIH45–46 G54W (F) and sCD4–183 (G) in log μg/ml, and the y-axis shows the percentage inhibition relative to infection without any antibody. The data shown are the means ± SEM of a representative experiment performed in triplicate. (H, I and J) IC50 values for VRC01 (H), NIH45–46 G54W (I) or sCD4–183 (J) are shown as mean ± SEM and each dot represents data obtained from cells derived from an independent donor. Two-tailed P values were calculated using unpaired (C and D) or paired (H, I and J) t-test in GraphPad Prism 5. * P<0.05, **: P < 0.01, ***: P < 0.0001, n.s.: not significant.

Article Snippet: Cells were blocked with 20% normal mouse serum, and CD169 expression was visualized with Alexa647-conjugated anti-CD169 mAb (AbD Serotec).

Techniques: Incubation, Staining, Isolation, Concentration Assay, Inhibition, Infection, Derivative Assay, Two Tailed Test

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: Capture of HIV-1 particles by CD169 leads to the formation of CD169 + VCCs in LPS-matured DCs. Lateral membrane movement of CD169-bound HIV-1 can result in accumulation of HIV-1 particles in plasma membrane microdomains in LPS-DCs. Multivalent interactions between multiple CD169 and HIV-1 particles and co-factor(s) recruitment might induce localized stress and strain to which the plasma membrane responds by forming invaginations. Arrow indicates lateral movement of CD169-bound HIV-1 particles into the VCC.

Article Snippet: Cells were blocked with 20% normal mouse serum, and CD169 expression was visualized with Alexa647-conjugated anti-CD169 mAb (AbD Serotec).

Techniques: Membrane, Clinical Proteomics

Journal: World Journal of Gastroenterology

Article Title: To screen or not to screen? Celiac antibodies in liver diseases

doi: 10.3748/wjg.v23.i5.776

Figure Lengend Snippet: Research on screening celiac disease in patients with primary biliar cholangitis

Article Snippet: According to Bizzaro et al[ ], 26.7% of PBC patients exhibited tTG positivity on performing at least one of six different ELISA tests (human recombinant, Eurospital; human recombinant, Pharmacia; human placenta, Euroimmun; human red blood cells, Inova; guinea pig liver, Eurospital; and guinea pig liver, Inova); however, a true association between PBC and CD was present in only 2% of patients who exhibited EmA positivity and showed histological patterns indicative of CD.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: World Journal of Gastroenterology

Article Title: To screen or not to screen? Celiac antibodies in liver diseases

doi: 10.3748/wjg.v23.i5.776

Figure Lengend Snippet: Research screening celiac disease in patients with autoimmune hepatitis

Article Snippet: According to Bizzaro et al[ ], 26.7% of PBC patients exhibited tTG positivity on performing at least one of six different ELISA tests (human recombinant, Eurospital; human recombinant, Pharmacia; human placenta, Euroimmun; human red blood cells, Inova; guinea pig liver, Eurospital; and guinea pig liver, Inova); however, a true association between PBC and CD was present in only 2% of patients who exhibited EmA positivity and showed histological patterns indicative of CD.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Animal Models and Experimental Medicine

Article Title: Elimination of GGTA1 , CMAH , β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

doi: 10.1002/ame2.12461

Figure Lengend Snippet: Construction and evaluation of 4KO piglets in xenotransplantation. (A) Schematic diagram of method for generating 4KO piglets. (B) Schematic of the 10 modified alleles. We generated the 4KO edits using CRISPR–Cas9 with gRNAs targeting the 2 copies of GGTA1 , 2 copies of CMAH , 4 copies of β4GalNT2 and 2 copies of the CIITA . (C) Deficiency of GGTA1 , CMAH , β4GalNT2 , and CIITA can impair the antigens of αGal, Neu5Gc, SDa, and SLA class II molecules, respectively. Flow cytometry was used to verify the deficiency of αGal, Neu5Gc, SDa, and SLA class II‐DR molecules at the cellular level in 4KO piglets. PBMCs of 4KO pigs had negative population cells (WT = 3, 4KO = 3). (D) Immunofluorescence results showed that αGal, Neu5Gc, SDa, and SLA class II molecules were not expressed in kidney tissue of 4KO piglets, and GGTA1 , CMAH , β4GalNT2 , and CIITA are genes were used to knockout the antigen of αGal, Neu5Gc, SDa and SLA class II molecules, respectively. Scale bar: 20 μm. (E) Verification by HE staining of the viability of 4KO tissue after gene editing; Scale bar: 100 μm.

Article Snippet: Stimulated pig cell samples were 2 × 10 5 WT or 4KO pig PBMCs treated with 50 μg/mL Mitomycin C (S8146, Selleck, Houston, TX, USA) for 20 min at 37°C to block their capacity for proliferation.

Techniques: Modification, Generated, CRISPR, Flow Cytometry, Immunofluorescence, Knock-Out, Staining

Journal: Animal Models and Experimental Medicine

Article Title: Elimination of GGTA1 , CMAH , β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

doi: 10.1002/ame2.12461

Figure Lengend Snippet: Changes in the pig immune system induced by CIITA deficiency (WT = 3, 4KO = 3): (A) Percentage of the population of CD3 in WT and 4KO. (B) Percentage of the population of γδT in WT and 4KO. (C) 4KO CD3 T cells are not significantly different from WT. (D) 4KO γδT are not significantly different from WT. (E) Percentage of the population of CD4+ T and CD8+ T in WT and 4KO. (F) 4KO CD4+ T cells significantly decreased relative to WT; 4KO CD8+ T cells significantly increased relative to WT. (G) B cell population of WT and 4KO PBMCs.( n = 3). (H) No significant difference between WT and 4KO in the population of B cells. (I) Percentage of 4KO NK cell population significantly decreased relative to WT. (J) NK cell population of WT and 4KO PBMCs. ** p < 0.01; * p < 0.05.

Article Snippet: Stimulated pig cell samples were 2 × 10 5 WT or 4KO pig PBMCs treated with 50 μg/mL Mitomycin C (S8146, Selleck, Houston, TX, USA) for 20 min at 37°C to block their capacity for proliferation.

Techniques:

Journal: Animal Models and Experimental Medicine

Article Title: Elimination of GGTA1 , CMAH , β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

doi: 10.1002/ame2.12461

Figure Lengend Snippet: (A) Verifying the function of CIITA deficiency, T cell proliferation was analyzed based on dilution of the proliferation dye CSFE with each cell division, Proliferation of human CD4+ T cells is depicted as percent of divided cell (WT = 3; 4KO = 3). (B) 4KO has lower immunogenicity to lead human CD4+ T proliferation than WT (WT = 3; 4KO = 3). (C) Verifying the Serum‐mediated antibody binding reaction of WT and 4KO, compared with WT, 4KO show significantly reduced binding to human IgG (a) and IgM (WT = 8; 4KO = 8). (D) Verifying the Human complement‐dependent cytotoxicity of WT and 4KO, 4KO show significantly lower antibody‐dependent complement cytotoxicity compared with WT (WT = 5; 4KO = 5). Unpaired two‐tailed t ‐test; ** p < 0.01; * p < 0.05).

Article Snippet: Stimulated pig cell samples were 2 × 10 5 WT or 4KO pig PBMCs treated with 50 μg/mL Mitomycin C (S8146, Selleck, Houston, TX, USA) for 20 min at 37°C to block their capacity for proliferation.

Techniques: Immunopeptidomics, Binding Assay, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Calcium-Sensing Receptor Is Involved in Follicle-Stimulating Hormone-Induced Cumulus Expansion in in vitro Cultured Porcine Cumulus-Oocyte Complexes

doi: 10.3389/fcell.2021.625036

Figure Lengend Snippet: Expression and localization of CASR in porcine cumulus cells. Cumulus-oocyte complexes (COCs) or oocytes in the germinal vesicle (GV) stage (intact nucleus) were used immediately after collection. Identification of CASR protein (A) and mRNA (B) in porcine cumulus cells and denuded oocytes; negative control without cDNA. (C) Immunofluorescence results showed that CASR protein (green) was expressed in cumulus cells of porcine COCs. Chromatin was stained with DAPI (blue). Negative controls show no staining for CASR. In the negative control group, the first antibody was replaced by 2% BSA, with the other steps being the same. Scale bar = 50 μm. CASR, Calcium-Sensing Receptor.

Article Snippet: Then they were permeabilized in Dulbecco’s phosphate buffered saline (DPBS, Gibco, Grand Island, NY) containing 1% Triton X-100 for 1 h at 37°C and blocked in DPBS containing 2% BSA at 37°C for 30 min. COCs were incubated with anti-CASR primary antibody (sc-32181, Santa Cruz Biotechnology, Santa Cruz, CA, United States) diluted 1:25 in blocking buffer at 37°C for 2 h. After washing three times, samples were incubated with DyLight TM 488-conjugated AffiniPure Rabbit anti-Goat IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:35 in blocking buffer at 37°C for 1 h (in the dark).

Techniques: Expressing, Negative Control, Immunofluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Calcium-Sensing Receptor Is Involved in Follicle-Stimulating Hormone-Induced Cumulus Expansion in in vitro Cultured Porcine Cumulus-Oocyte Complexes

doi: 10.3389/fcell.2021.625036

Figure Lengend Snippet: Relative expression of CASR mRNA in porcine cumulus cells from COCs matured in the presence or absence of FSH for 24 h by qRT-PCR. Data are presented as means ± SEM (* p < 0.05) from three independent experiments. (*) indicates significance between groups. Significance was determined by independent t -test. CASR, Calcium-Sensing Receptor.

Article Snippet: Then they were permeabilized in Dulbecco’s phosphate buffered saline (DPBS, Gibco, Grand Island, NY) containing 1% Triton X-100 for 1 h at 37°C and blocked in DPBS containing 2% BSA at 37°C for 30 min. COCs were incubated with anti-CASR primary antibody (sc-32181, Santa Cruz Biotechnology, Santa Cruz, CA, United States) diluted 1:25 in blocking buffer at 37°C for 2 h. After washing three times, samples were incubated with DyLight TM 488-conjugated AffiniPure Rabbit anti-Goat IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:35 in blocking buffer at 37°C for 1 h (in the dark).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Calcium-Sensing Receptor Is Involved in Follicle-Stimulating Hormone-Induced Cumulus Expansion in in vitro Cultured Porcine Cumulus-Oocyte Complexes

doi: 10.3389/fcell.2021.625036

Figure Lengend Snippet: Effects of CASR activation or inhibition on cumulus cell expansion during IVM of porcine COCs. COCs were cultured in base IVM medium supplemented with FSH (0.01 U/ml), and/or the CASR agonist NPSR-568 (5 or 10 μM, A) and/or the CASR inhibitor NPS2390 (10 or 20 μM, I) for 24 h. FSH group was taken as a unit. Statistically significant differences are examined by independent t -test. Data are expressed as mean ± SEM from three independent experiments (* p < 0.05). CASR, Calcium-Sensing Receptor; A, CASR agonist NPSR-568; I, CASR inhibitor NPS2390.

Article Snippet: Then they were permeabilized in Dulbecco’s phosphate buffered saline (DPBS, Gibco, Grand Island, NY) containing 1% Triton X-100 for 1 h at 37°C and blocked in DPBS containing 2% BSA at 37°C for 30 min. COCs were incubated with anti-CASR primary antibody (sc-32181, Santa Cruz Biotechnology, Santa Cruz, CA, United States) diluted 1:25 in blocking buffer at 37°C for 2 h. After washing three times, samples were incubated with DyLight TM 488-conjugated AffiniPure Rabbit anti-Goat IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:35 in blocking buffer at 37°C for 1 h (in the dark).

Techniques: Activation Assay, Inhibition, Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Calcium-Sensing Receptor Is Involved in Follicle-Stimulating Hormone-Induced Cumulus Expansion in in vitro Cultured Porcine Cumulus-Oocyte Complexes

doi: 10.3389/fcell.2021.625036

Figure Lengend Snippet: Relative expression of genes related to cumulus cell expansion in porcine COCs cultured under different conditions. COCs were cultured in medium supplemented with the CASR agonist NPSR-568 (10 μM, A–C ) or inhibitor NPS2390 (20 μM, D–F ) in the presence or absence of FSH for 24 h. ACTIN mRNA was used as internal control. HAS2 : hyaluronan synthase 2; PTGS2: prostaglandin-endoperoxide synthase 2; TNFAIP6: tumor necrosis factor a-induced protein 6; CASR: Calcium-Sensing Receptor. The data represent means ± SEM (* p < 0.05) from at least two independent experiments. (*) indicates significance between groups; (ns) indicates no significance between groups. Significance was determined by Kruskal-Wallis test and Mann-Whitney U -test (A–C) or two-tailed t -tests (E,F) .

Article Snippet: Then they were permeabilized in Dulbecco’s phosphate buffered saline (DPBS, Gibco, Grand Island, NY) containing 1% Triton X-100 for 1 h at 37°C and blocked in DPBS containing 2% BSA at 37°C for 30 min. COCs were incubated with anti-CASR primary antibody (sc-32181, Santa Cruz Biotechnology, Santa Cruz, CA, United States) diluted 1:25 in blocking buffer at 37°C for 2 h. After washing three times, samples were incubated with DyLight TM 488-conjugated AffiniPure Rabbit anti-Goat IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:35 in blocking buffer at 37°C for 1 h (in the dark).

Techniques: Expressing, Cell Culture, Control, MANN-WHITNEY, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome

doi: 10.3389/fcell.2020.00440

Figure Lengend Snippet: MYH6 -R443P VAR-inserted iPSC-CMs recapitulate phenotypes of patient-specific iPSC-CMs. (A) Sequences at the MYH6- R443 locus in VAR-inserted lines. * is R443 locus and * is locus for silencing mutation. (B) Scheme for differentiating CMs using small molecule Gsk3 inhibitor (CHIR99021) with Activin-A and Wnt inhibitor (IWP). (C) Representative immunostaining of VAR-inserted iPSC-CMs with MF20 at D12. (D) Representative flow cytometry of cardiac troponin T (cTnT) positive cells in CRISPRed iPSC-CMs at D12. (E) Flow cytometry of cells cultured in parallel with those in (D) , showing % cTnT-positive cells in VAR-inserted iPSC-CMs at D10-15. Data were compiled from 16 replicates in each cell type and 5 separate experiments. (F) Sarcomeres are dysmorphic in VAR-introduced iPSC-CMs as compared to WT CC . α-actinin immunostaining (red) is seen in high density cultures at D50. (G) Comparative sarcomere organization in single cell iPSC-CMs isolated after sub-culturing at a low density at D69-D73. (H) % of cells with normal organized sarcomeres in both VAR-inserted iPSC-CMs, +/VAR and VAR/VAR in parallel to those in (G) . WTcc, CRISPRed control wild-type; +/VAR, MYH6- R443P heterozygous inserted; VAR/VAR, MYH6- R443P homozygous inserted. Values are means ± SE. Student’s t -test (two-tailed, equal variance), *** P < 0.0005.

Article Snippet: The iPSC-CMs and tissues were co-immunostained with anti-sarcomeric α-actinin (AF594) (Abcam, Cambridge, MA, United States) to image sarcomere organization, and anti-MHC monoclonal MF20 (AF488) (DSHB, University of Iowa, Iowa City, IA, United States) to inform myocyte identity.

Techniques: Mutagenesis, Immunostaining, Flow Cytometry, Cell Culture, Isolation, Control, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome

doi: 10.3389/fcell.2020.00440

Figure Lengend Snippet: MYH6 -R443P VAR-corrected iPSC-CMs rescues the phenotype of proband iPSC-CMs. (A) Sequences at the MYH6- R443P locus in corrected line. * is the R443 locus and * is the locus for a silencing mutation. (B) Representative immunostaining of VAR-corrected iPSC-CMs with MF20 (red) at D10. (C) Representative flow cytometry of cardiac troponin T (cTnT) positive cells in corrected iPSC-CMs at D12. (D) Flow cytometry of cells cultured in parallel with those in (C) , showing % cTnT-positive cells at D10. Each dot is a technical replicate (Probandcc n = 2 and corrected WT n = 4). (E) Sarcomeres appear more “normal” in corrected WT iPSC-CMs compared to Probandcc iPSC-CMs. α-actinin immunostaining is seen in red and GATA4 in green in high density cultures at D63. Probandcc, CRISPRed control proband; corrected WT, CRISPRed and corrected MYH6 -R443P variant.

Article Snippet: The iPSC-CMs and tissues were co-immunostained with anti-sarcomeric α-actinin (AF594) (Abcam, Cambridge, MA, United States) to image sarcomere organization, and anti-MHC monoclonal MF20 (AF488) (DSHB, University of Iowa, Iowa City, IA, United States) to inform myocyte identity.

Techniques: Mutagenesis, Immunostaining, Flow Cytometry, Cell Culture, Control, Variant Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome

doi: 10.3389/fcell.2020.00440

Figure Lengend Snippet: Dysmorphic sarcomeres were observed in atrial tissues from HLHS patients with MYH6 variants, but not in ventricles. Sarcomeric α-actinin is in red, MF20 in green and dapi in blue. (A–D) Atrial tissues from HLHS free patients without MYH6 variants. (E–H) Atrial tissues from HLHS patients without MYH6 variants. (I–L) Atrial tissues from HLHS patients with MYH6 variants. (M–P) Ventricular tissues from HLHS patients without MYH6 variants. (Q,R) Ventricular tissues from HLHS patients with MYH6 variants.

Article Snippet: The iPSC-CMs and tissues were co-immunostained with anti-sarcomeric α-actinin (AF594) (Abcam, Cambridge, MA, United States) to image sarcomere organization, and anti-MHC monoclonal MF20 (AF488) (DSHB, University of Iowa, Iowa City, IA, United States) to inform myocyte identity.

Techniques:

Journal: Journal of Hypertension

Article Title: EPHA4 regulates vascular smooth muscle cell contractility and is a sex-specific hypertension risk gene in individuals with type 2 diabetes

doi: 10.1097/hjh.0000000000001948

Figure Lengend Snippet: FIGURE 1 LocusZoom plots of log10 P values and r2 values of EPHA4 single nucleotide variants in regions analyzed for association with hypertension and rates of recombination in the ADVANCE study. Symbols (circles and diamonds) and the left-hand vertical axis illustrate the log10 P values of SNPs. Lines and the right-hand vertical axis represent recombination rates in the chromosomal regions concerned. Symbol colors represent r2 values. The horizontal axis indicates position in chromosomes. (a) Males-only samples. (b) Females-only samples. ADVANCE, Action in Diabetes and Vascular Disease: Preterax and Diamicron-MR Controlled Evaluation; SNPs, single nucleotide polymorphisms.

Article Snippet: In some experiments, cell culture plates were coated with goat antimouse EPHA4 antibody (aEPHA4-Ab, AF641; R&D Systems, Minneapolis, Minnesota, USA; 2 mg/ml during coating), normal goat IgG (NGIgG; 2 mg/ml during coating), recombinant mouse EPHA4 tagged with human IgG Fc (EPHA4-Fc, 641-A4; R&D Systems; 10 mg/ml during coating) or normal human IgG (NHIgG; 10 mg/ml during coating) overnight at 4 8C with gentle shaking.

Techniques:

Journal: Journal of Hypertension

Article Title: EPHA4 regulates vascular smooth muscle cell contractility and is a sex-specific hypertension risk gene in individuals with type 2 diabetes

doi: 10.1097/hjh.0000000000001948

Figure Lengend Snippet: FIGURE 2 EPHA4 protein expression in mouse vascular smooth muscle cells according to immunofluorescence microscopy. Wild type VSMCs from male C57BL/6 mice were cultured for 4–5 days and stained with goat anti-EPHA4 Ab (a) or normal goat IgG (b) (both at 15 mg/ml), followed by FITC-conjugated donkey antigoat secondary Ab (1.5 mg/ml, in pseudo green). For a-actin staining (c), the cells were permeabilized and stained with mouse antihuman a-actin Ab (200 ng/ml), followed by rhodamine- conjugated goat antimouse Ab (1.4 mg/ml, in pseudo-red). Nuclei were identified by DAPI staining (in pseudo-blue). The images in (a) and (c) are merged in (d) to show EPHA4 and a-actin staining at the same time. The experiments were repeated three times, and representative results are shown. DAPI, 40,6-diamidino-2-phenylindole; FITC, fluorescein-5-isothiocyanate; VSMC, vascular smooth muscle cell.

Article Snippet: In some experiments, cell culture plates were coated with goat antimouse EPHA4 antibody (aEPHA4-Ab, AF641; R&D Systems, Minneapolis, Minnesota, USA; 2 mg/ml during coating), normal goat IgG (NGIgG; 2 mg/ml during coating), recombinant mouse EPHA4 tagged with human IgG Fc (EPHA4-Fc, 641-A4; R&D Systems; 10 mg/ml during coating) or normal human IgG (NHIgG; 10 mg/ml during coating) overnight at 4 8C with gentle shaking.

Techniques: Expressing, Immunofluorescence, Microscopy, Cell Culture, Staining

Journal: Journal of Hypertension

Article Title: EPHA4 regulates vascular smooth muscle cell contractility and is a sex-specific hypertension risk gene in individuals with type 2 diabetes

doi: 10.1097/hjh.0000000000001948

Figure Lengend Snippet: FIGURE 3 Expression of EPHA4 in human mesenteric arteries. (a) EPHA4 mRNA expression in VSMCs from human mesenteric arteries according to reverse transcription real time-quantitative PCR (RT-qPCR). Adventitia and endothelium of human mesenteric arteries were stripped off, and EPHA4 mRNA expression in the remaining VSMCs was measured by RT-qPCR. Samples were in triplicate in RT-qPCR measurement. EPHB6 mRNA expression was included as a reference to show the relative expression level of EPHA4. The results of three independent experiments are pooled and are expressed as mean standard error (SE) of ratios of EPH versus b-actin signals. (b) EPHA4 protein expression in human mesenteric arteries according to immunofluorescence microscopy. Human mesenteric arteries were cryo-sectioned, and stain with rabbit antihuman EPHA4 Ab (a and c; pseudo-red). Normal rabbit IgG was used as isotypic control Ab (b and d). Nuclei were stained with DAPI (pseudo-blue). Both endothelium (arrow) and VSMC stainings are shown in (a), and VSMC staining is shown in (b). Representative images of similar ones obtained in three independent experiments are shown. DAPI, 40,6-diamidino-2-phenylindole; VSMC, vascular smooth muscle cell; RT-qPCR, reverse transcription real time-quantitative PCR.

Article Snippet: In some experiments, cell culture plates were coated with goat antimouse EPHA4 antibody (aEPHA4-Ab, AF641; R&D Systems, Minneapolis, Minnesota, USA; 2 mg/ml during coating), normal goat IgG (NGIgG; 2 mg/ml during coating), recombinant mouse EPHA4 tagged with human IgG Fc (EPHA4-Fc, 641-A4; R&D Systems; 10 mg/ml during coating) or normal human IgG (NHIgG; 10 mg/ml during coating) overnight at 4 8C with gentle shaking.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining, Control

Journal: Journal of Hypertension

Article Title: EPHA4 regulates vascular smooth muscle cell contractility and is a sex-specific hypertension risk gene in individuals with type 2 diabetes

doi: 10.1097/hjh.0000000000001948

Figure Lengend Snippet: FIGURE 4 EPHA4 forward signaling regulates vascular smooth muscle cell contractility in concert with estrogen. VSMCs from male and female C57BL/6J mice (as indicated) were cultured in wells coated with goat antimouse EPHA4 Ab or control normal goat IgG (NGIgG; both at 2 mg/ml during coating) for 4 days, in the absence or presence of estrogen (100 ng/ml) or testosterone (6.5 ng/ml). The cells were then stimulated with (R)-(-)-phenylephrine hydrochloride (20 mmol/l) and imaged continuously for 15 min at one frame per minute in a controlled environment (37 8C in 5% CO2). Images of 15 or more cells were selected randomly, with their length measured at the time point indicated. The results are expressed as means SEM of contraction percentage (% contraction), calculated as follows: % contraction ¼ 100 (cell length at time 0 cell length at time X)/cell length at time 0. P less than 0.05; P less than 0.01: two-tailed Student’s t-tests. The experiments were repeated three times and representative results are reported. (a and b) Reduced contractility of male (a) and female (b) VSMCs when stimulated with solid-phase anti-EPHA4 Ab. (c) Lower contractility of female VSMCs stimulated with solid-phase anti-EPHA4 Ab compared with that of their male counterparts. (d) Similar contractility of male and female VSMCs without solid-phase anti-EphA4 Ab stimulation. (e) Estrogen enhances the contractility lowering effect of solid-phase anti-EphA4 Ab in male VSMCs. (f) Testosterone has no effect on the contractility of female VSMCs stimulated by anti-EPHA4 Ab. (g and h) Estrogen (g) and testosterone (h) have no effect on VSMC contractility from male and female mice, respectively. VSMC, vascular smooth muscle cell.

Article Snippet: In some experiments, cell culture plates were coated with goat antimouse EPHA4 antibody (aEPHA4-Ab, AF641; R&D Systems, Minneapolis, Minnesota, USA; 2 mg/ml during coating), normal goat IgG (NGIgG; 2 mg/ml during coating), recombinant mouse EPHA4 tagged with human IgG Fc (EPHA4-Fc, 641-A4; R&D Systems; 10 mg/ml during coating) or normal human IgG (NHIgG; 10 mg/ml during coating) overnight at 4 8C with gentle shaking.

Techniques: Cell Culture, Control, Two Tailed Test

Journal: Journal of Hypertension

Article Title: EPHA4 regulates vascular smooth muscle cell contractility and is a sex-specific hypertension risk gene in individuals with type 2 diabetes

doi: 10.1097/hjh.0000000000001948

Figure Lengend Snippet: FIGURE 5 Effect of EPHA4 reverse signaling and knockdown in vascular smooth muscle cell contractility. (a and b) Reverse signaling from EPHA4 to EFNs does not affect VSMC contractility. VSMCs from male (a) and female (b) C57BL/6J mice were cultured in wells coated with EPHA4-Fc or NHIgG (both at 10 mg/ml for coating). (R)-(-)- phenylephrine hydrochloride (20 mmol/l)-stimulated contractility was ascertained after 4 to 5 days of culture. The experiments were repeated three times, and representative results are presented as means SEM of contraction percentage of 15 randomly selected cells. No significant difference between EPHA4-Fc and NHIgG-stimulated cells is evident (two-tailed Student’s t-test). (c–h) Increased contractility in female but not male VSMCs after EPHA4 knockdown. VSMCs from male and female C57BL/6J mice (as indicated) were cultured in uncoated wells. After 48 h, the cells were cultured in antibiotic-free media and transfected with Epha4 siRNA (two pairs of siRNA each at 20 nmol/l) or control siRNA (40 nmol/l). After another 48 h, Epha4 mRNA levels in Epha4 siRNA-transfected and control siRNA-transfected cells were quantified by RT-qPCR. Pooled results from three independent experiments (mean SD of ratios of Epha4 signals versus b-actin signals) are shown in (c) and (d). EPHA4 protein knockdown by siRNA in male and female VSMCs were shown in (e) and (f) according to immunofluorescence, in which means and SD of immunofluorescence intensity of more than 15 randomly selected cells in a representative experiment are presented. Background signal (isotypic Ab staining) was deducted from the anti-EPHA4 Ab staining signals. (R)-(- )-phenylephrine hydrochloride (20 mmol/l)-triggered contractility of these siRNA-transfected cells was measured at 48 h posttransfection (g and h). The experiments were repeated three times, and representative results are shown. The result was expressed as means SEM of contraction percentage. P < 0.05; P < 0.01: two-tailed Student’s t-test. EFNs, ephrins; VSMC, vascular smooth muscle cell.

Article Snippet: In some experiments, cell culture plates were coated with goat antimouse EPHA4 antibody (aEPHA4-Ab, AF641; R&D Systems, Minneapolis, Minnesota, USA; 2 mg/ml during coating), normal goat IgG (NGIgG; 2 mg/ml during coating), recombinant mouse EPHA4 tagged with human IgG Fc (EPHA4-Fc, 641-A4; R&D Systems; 10 mg/ml during coating) or normal human IgG (NHIgG; 10 mg/ml during coating) overnight at 4 8C with gentle shaking.

Techniques: Knockdown, Cell Culture, Two Tailed Test, Transfection, Control, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Liver X Receptor β Controls Hepatic Stellate Cell Activation via Hedgehog Signaling

doi: 10.1101/577833

Figure Lengend Snippet: (A) a, Images of freshly isolated, quiescent HSCs cultured for 24 h; HSCs appear as round, phase-dense cells containing refractile lipid droplets. b, Oil red O staining of HSCs cultured for 24 h; HSCs displayed abundant garland-like lipid droplets in the cytoplasm. c, Immunohistochemical staining of HSCs cultured for 24 h; HSCs were positive for the quiescent HSC marker GFAP. d, Immunohistochemical staining of HSCs cultured for 168 h; HSCs were positive for the activated HSC marker Acta2. (B) Relative mRNA expression of collagen I and Acta2 during HSC activation. (C) Relative mRNA expression of LXRs and downstream target genes Abca1 and Srebp-1c during HSC activation. (D) Protein expression of collagen I, Acta2, LXRα, LXRβ, Srebp-1c and Abca1 during HSC activation was investigated by western blotting, using β-actin as a loading control. DAPI was used to stain the nucleus. n≥3, *P<0.05, **P<0.01. Scale bars, 50 μm.

Article Snippet: Further, the purities of primary cells were tested using glial fibrillary acidic protein (GFAP) (quiescent HSC marker; 1:100; 16825-1-AP, Proteintech Group, China) or Acta2 (activated HSC marker; 1/100; ab7817; Abcam, Cambridge, UK) for HCSs, and cytokeratin 18 (HC marker; 1:100; 10830-1-AP, Proteintech Group) for HCs ( ).

Techniques: Isolation, Cell Culture, Staining, Immunohistochemical staining, Marker, Expressing, Activation Assay, Western Blot

Journal: Brain

Article Title: Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease

doi: 10.1093/brain/awz364

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: , Cy2 (anti-mouse, rat, rabbit, goat IgG) , Donkey , 1:200 , Jackson ImmunoResearch Laboratories , .

Techniques: Flow Cytometry

Journal: Brain

Article Title: Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease

doi: 10.1093/brain/awz364

Figure Lengend Snippet: Enhanced surface recognition and uptake of diverse amyloid-β42 conformers by ACE10 macrophages. (A) Design and timeline of in vitro. Bone marrow was isolated from young wild-type (WT) and ACE10 mice (8–12 weeks old) and cultured for 6 days in MCSF-enriched media, generating primary macrophage cultures (MφWT and MφACE10, respectively). On Day 7, Mφ were either unstimulated (Un) or stimulated by one of the three amyloid-β forms: fibrils (fAβ42), prion rod-like structures (pAβ42), or cross-linked oligomers (oAβ42). MφACE10 were analysed and compared to control MφWT for cell surface amyloid-β42 binding (pre-chilled on ice; binding – 0 min) and receptor expression as well as uptake of amyloid-β42 species at incremental time points from 5 min up to 60 min (incubated at 37°C; uptake: 5, 15, 30 min). Cells and media were isolated and further analysed using various methods including ICC, flow cytometry (FC), and quantitative protein analysis. (B) Forms of non-cross-linked (non-XL) and cross-linked (oAβ) amyloid-β42. Oligomers as large as an octamer are observed. (C) Negative stain TEM micrographs of amyloid-β42 assemblies. Left: Fibrillar amyloid-β (fAβ42) displayed long fibrils of ˜10 nm diameter with a twisted morphology typically comprising two smaller filaments. Middle: Prion rod-like amyloid-β42 (pAβ42) displayed short (<300 nm) straight rods comprising multiple filaments (blue arrows). Narrower (˜5 nm) rod-like structures appeared to comprise twisted double filaments (black arrows). The assembly indicated by the red arrow is shown in the inset at higher magnification. Right: Cross-linked oligomers (oAβ42) displayed mainly quasi-spherical assemblies (white arrows) with diameters of 5–8 nm. The assembly indicated by the red arrow is shown in the inset at higher magnification. A rare rod-like structure (yellow asterisk) was also observed. Scale bars = 100 nm or 50 nm (insets). (D) Representative fluorescence micrograph of MφACE10 prestimulated with fibril amyloid-β42 on ice (at the 0-min time point) and immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Abundance of co-surface labelling of Scara1+ and fibril amyloid-β42 (yellow spots) on the MφACE10 cell body and processes was detected, indicating large amounts of fibril amyloid-β42 bound to surface Scara1. (E) Quantitative ICC analysis of a 6E10+ immunoreactive (IR) area for cell surface binding of the three amyloid-β42 species (250 nM each) at the 0-min time point in MφACE10 versus MφWT. (F) Quantitative ICC analysis of surface Scara1 expression in ACE10 versus wild-type Mφ that were either untreated or in the presence of different amyloid-β42 forms at the 0-min time point. (G) Quantitative ICC analysis of TREM2 expression in ACE10 versus wild-type Mφ following prestimulation by the different forms of amyloid-β42 (250 nM each). (H) Representative fluorescence micrograph of MφWT and MφACE10 cells that were incubated for 5 min with 250 nM fibril amyloid-β42 and later immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Amyloid-β42 fibrils are seen in intracellular vesicles within these phagocytic cells (white arrow). (I) Panel of representative fluorescence micrographs of MφWT and MφACE10 cells following a 15-min incubation with three different amyloid-β42 forms immunolabelled for scavenger receptor type B CD36 (purple), human amyloid-β (6E10; green), and nuclei (blue). (I’) Separated single channel (cy2) for intracellular amyloid-β42 uptake. (J) Quantitative ICC analysis of CD36 expression (μm2/cell number) in ACE10 versus wild-type Mφ that were either untreated or exposed to different amyloid-β42 forms (250 nM) for 15 min. (K) Quantitative ICC analysis of 6E10+ immune-reactive area (μm2/cell number) for intracellular uptake of the three amyloid-β42 species (250 nM) for 15 min by MφACE10 versus MφWT. (L and M) Quantitative ICC analysis of (L) CD36 expression and (M) amyloid-β uptake by untreated versus ACE-inhibitor pretreated (1 μM lisinopril overnight in RPMI media + 0.3% BSA) MφACE10, following exposure to 100 nM fibril amyloid-β42 for either 0, 5 or 30 min. (N–Q) Individual correlation analysis of the following: (N and O) surface fibril amyloid-β42 and Scara1 immunoreactive area at the binding time point in (N) MφWT and (O) MφACE10 (n =15 wells); (P and Q) surface fibril/oligomer amyloid-β42 and CD36 expression in MφACE10 following a 15-min exposure to either fibril amyloid-β42 or oligomer amyloid-β42. (R) Representative peak analysis in percentage mean fluorescence intensity (MFI) of MφACE10 uptake of HiLyte Fluor 647-labelled fibril amyloid-β42 at the 0-, 5-and 15-min time points. (S) Representative flow cytometry images of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI at the 15-min time point by F4/80+-MφWT versus -MφACE10. (T and U) Quantitative flow cytometry analysis of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI uptake at the 0-, 5- and 15-min time points by (T) MφWT or MφACE10 and by (U) untreated and lisinopril pretreated MφACE10. The group mean ± SEM as well as the fold reduction or increase and percentage decrease between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s or Sidak’s post-test, while asterisks in parentheses signify a two-group comparison using the unpaired two-tailed Student t-test.

Article Snippet: , Cy2 (anti-mouse, rat, rabbit, goat IgG) , Donkey , 1:200 , Jackson ImmunoResearch Laboratories , .

Techniques: In Vitro, Isolation, Cell Culture, Control, Binding Assay, Expressing, Incubation, Flow Cytometry, Staining, Fluorescence, Comparison, Two Tailed Test