Journal: Amino Acids

Article Title: Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells

doi: 10.1007/s00726-023-03263-8

Figure Lengend Snippet: Protein expression and G6Pase enzymatic activity of hG6Pase-α variants directed by hG6PC mRNA constructs. A Topological analysis of hG6Pase-α shows a nine-transmembrane spanning ER enzyme. Black dots: locations of Q139, Q247, and S298 for targeted substitutions. Blue dots: residues directly involved in enzymatic activity. B and C protein expression ( B ) and G6Pase enzymatic activity ( C ) of hG6Pase-α variants encoded by hG6PC mRNA constructs encoding wild-type protein (WT) or protein variants containing a point mutation as indicated. D and E protein expression ( D ) and G6Pase enzymatic activity ( E ) of WT and the S298C hG6Pase-α variants encoded by two different codons at the mutated site. The mRNA constructs were transfected into HeLa cells and examined for protein expression and G6Pase activity in cell lysates at 48-h post-transfection, as described under “ ”. For protein expression analysis, the quantified signals from the hG6Pase-α variants were also normalized by Erp72, a house-keeping ER marker protein. Data were shown as percentage of wild-type (WT) group and presented as mean ± SEM of n = 3 samples from independent transfections. * p < 0.05; ** p < 0.01; *** p < 0.00, **** p < 0.0001. ns non-significant

Article Snippet: HeLa cells were plated in 96-well, glass bottom plates (655,892, Greiner Bio-One) using recommended culturing conditions, at a density of 15,000 cells per well.

Techniques: Expressing, Activity Assay, Construct, Mutagenesis, Transfection, Marker

Journal: Amino Acids

Article Title: Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells

doi: 10.1007/s00726-023-03263-8

Figure Lengend Snippet: Kinetic analysis of protein expression ( A-D, F ) and G6Pase enzymatic activity ( E ) of wild-type (WT) hG6Pase-α and its variants harboring S298C or S298P mutation in mammalian cells transfected with hG6PC mRNA constructs. The mRNA constructs were transfected into HeLa cells and examined for protein expression and G6Pase activity in cell lysates at different time (2 to 72 h) post-transfection. Panel A represents a western blotting analysis of wild-type hG6Pase-α and variants harboring the S298C or S298P mutations, which migrated as two polypeptides (non-glycosylated or glycosylated), especially at the time points between 6 and 48 h, post-transfection. For protein expression analysis in panels B to D , the quantified signals from the hG6Pase-α variants were normalized by beta-actin. Panel E shows the G6Pase enzymatic activity in cell lysates prepared at different time points post-transfection. F kinetic analysis of subcellular expression and localization of wild-type (WT) hG6Pase-α and its variants harboring S298C or S298P. HeLa cells were transfected with mRNA constructs and analyzed for subcellular expression of hG6Pase-α and variants with immune-cytofluoresence-based confocal microscopy. Green: hG6Pase-α and its variants, Red: Calnexin, an ER marker. The cells were also counterstained with DAPI (blue) for nucleus visualization. All samples were stained for hG6Pase-α, Calnexin, and DAPI. Scale bars are 50 µm. All quantitative data are presented as mean ± SEM of n = 3 samples from independent transfections. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. WT at the same time point. Data shown on panel F are representative of at least two independent experiments. Hrs hours, UT untransfected

Article Snippet: HeLa cells were plated in 96-well, glass bottom plates (655,892, Greiner Bio-One) using recommended culturing conditions, at a density of 15,000 cells per well.

Techniques: Expressing, Activity Assay, Mutagenesis, Transfection, Construct, Western Blot, Confocal Microscopy, Marker, Staining

Journal: Amino Acids

Article Title: Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells

doi: 10.1007/s00726-023-03263-8

Figure Lengend Snippet: Protein expression ( A and C ) and G6Pase enzymatic activity ( B and D ) of wild-type (WT) hG6Pase-α and its variants targeted for selective substitution of cysteine. The mRNA constructs were transfected into HeLa cells and examined for protein expression of glycosylated hG6Pase-α and its variants and G6Pase activity in cell lysates at 24-h post-transfection, as described under “ ”. For protein expression analysis, the quantified signals from the glycosylated hG6Pase-α or its variants were also normalized by Erp72. Data were shown as percentage of WT group and presented as mean ± SEM of n = 3 samples from independent transfections. * p < 0.05; ** p < 0.01; *** p < 0.001; *** *p < 0.0001. ns non-significant

Article Snippet: HeLa cells were plated in 96-well, glass bottom plates (655,892, Greiner Bio-One) using recommended culturing conditions, at a density of 15,000 cells per well.

Techniques: Expressing, Activity Assay, Construct, Transfection

Journal: Amino Acids

Article Title: Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells

doi: 10.1007/s00726-023-03263-8

Figure Lengend Snippet: Effect of proteasome inhibitor, lactacystin, on protein expression of hG6Pase-α and its variants. HeLa cells were first transfected with mRNA constructs. At 6 h post-transfection, medium was replaced with that containing vehicle (0.1%, v/v, DMSO) or lactacystin at 10 µM and the cells were incubated for additional 16 h before they were collected for protein expression analysis. The graph shows the analysis of glycosylated hG6Pase-α and its variants. Data were presented as mean ± SEM of n = 3 samples from independent transfections. ** p < 0.01; *** p < 0.001 vs. vehicle. ns non-significant

Article Snippet: HeLa cells were plated in 96-well, glass bottom plates (655,892, Greiner Bio-One) using recommended culturing conditions, at a density of 15,000 cells per well.

Techniques: Expressing, Transfection, Construct, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Induction of mtRNA leakage by IFN-α-100 and ICs treatment. BMDMs (2 × 10 6 ) were stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h, and the levels of cytosolic mtRNA were determined by measuring both the heavy chain and the light chain of several mtRNA genes as indicated by RT-qPCR ( A and C ) and confocal microscopy ( B and D ). The heavy and light chains of some mtRNA genes in the supernatant were determined in BMDMs treated with IFN-α (100 U/ml) or 10 µg/ml ICs for different periods of time ( E and F ). The levels of cytosolic mtRNA in BMDMs treated with anti-chicken egg albumin Abs, albumin (ovalbumin) or a combination of anti-albumin Abs and albumin (ICs) were determined ( G ). Each data point represents one mouse, and the values are fold changes relative to the mean value of the unstimulated controls determined by RT-qPCR ( A , C , E , F , and G ). Representative results from at least 3 independent experiments are shown ( B and D ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( A , C , E , and F ) and two-way ANOVA with the Holm-Šídák’s multiple comparisons test to compare differences among different treatments ( G ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ND, NADH dehydrogenase; CYTB, cytochrome b

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Quantitative RT-PCR, Confocal Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: IFN-α-100 and ICs treatment selectively induces mitochondrial pore opening. BMDMs (2 × 10 6 ) were stimulated with IFN-α (100 U/ml) ( A ) or 10 µg/ml ICs ( B ) for 24 h, and mPTP opening was measured by calcein AM staining, and the results were analyzed by flow cytometry as described in the Materials and Methods. Images of calcein AM staining were analyzed by confocal microscopy ( C ). The protein levels of ANT1 and ANT2 were determined ( D ). The oligomerization of VDAC1 ( E and F ) and BAX ( G and H ) with or without EGS, a cross-linking reagent used to stabilize oligomers in electrophoresis, was evaluated, and the results of the statistical analysis are presented individually ( E to H ). As a positive control for BAX oligomerization, the treatment with LPS (200 ng/ml) for 4 h and then ATP (4 mM) for 1 h ( G ) or TNF-α (20 ng/ml) plus cycloheximide (CHX; 1 µg/ml) for 3 h ( H ) was given. BMDMs (2 × 10 6 ) were pre-treated with BAI-1 (2 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. As positive controls, BMDMs were pre-treated with BAI-1 (2 µM) for 2 h and then stimulated with staurosporine (STS; 2 µM) or TNF-α (20 ng/ml) plus cycloheximide (CHX; 30 µg/ml) for 3 h. MtRNA release was measured by RT-qPCR ( I ). Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A and B ). For Western blotting, the ratio of protein levels was calculated by comparing the intensity of a protein of interest to that of a housekeeping protein or monomer of protein as indicated ( D to H ). The samples were derived from the same experiment, and both the gels and the blots were processed in parallel. In ( I ), the values are fold changes relative to the mean value of the controls determined in RT-qPCR. Statistical analysis was performed with an unpaired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ANT, adenine nucleotide translocase; Tri, trimer; Di, dimer; PDTC, pyrrolidine dithiocarbamate ammonium

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Staining, Flow Cytometry, Confocal Microscopy, Electrophoresis, Positive Control, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Inhibition of mitophagy does not block IFN-α-100 and ICs-induced mtRNA release. BMDMs (2 × 10 6 ) were stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h, and the intensity of mitophagy was determined by flow cytometry as described in the Materials and Methods section, and the statistics are presented ( A and B ). C The results of the confocal microscopic imaging studies. D and E show the inhibitory effects of Mdivi-1 (50 µM), a mitophagy inhibitor, on IFN-α-100 and ICs-induced mitophagy by flow cytometry. Mdivi-1 did not affect cytosolic mtRNA release, as determined by measuring ND1 and Co-2 mRNA expression in the cytosol ( F and G ). Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A , B , D , and E ). The values are fold changes relative to the mean value of the controls in RT‒qPCR ( F and G ). Representative results from at least 3 independent experiments are shown ( C ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( A and B ). Two-way ANOVA with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments ( D to G ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The arrows show the colocalization of mitophagy dye and lysosomal marker

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Inhibition, Blocking Assay, Flow Cytometry, Imaging, Expressing, Marker

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Inhibition of mPTP opening and VDAC1 oligomerization attenuates IFN-α-100 and ICs-induced mtRNA release and downstream signaling. BMDMs (2 × 10 6 ) were pretreated with cyclosporin A (5 µM) or VBIT-12 (80 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. The intensity of mPTP opening ( A and D ), mtRNA release ( B , E , H , and K ), mRNA levels of several inflammatory genes ( C , F , I , and L ), and VDAC1 oligomerization ( G and J ) were measured as described in Materials and Methods. Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A and D ). The values are fold changes relative to the mean value of the controls as determined by RT-qPCR ( B , C , E , F , H , I , K , and L ). Representative results from at least 3 independent experiments are shown ( G and J ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( C , F , I , and L ). Two-way ANOVA with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments ( A , B , D , E , H , and K ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. RIG1, retinoic acid-inducible gene I; ISG15, interferon-stimulated gene 15; IL, interleukin; MDA5, melanoma differentiation-associated protein 5; OAS, 2′-5′-oligoadenylate synthetase; Tri, trimer; Di, dimer

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Inhibition, Flow Cytometry, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Blockade of mitochondrial calcium overload inhibits IFN-α-100 and ICs-induced mtRNA release. BMDMs (2 × 10 6 ) were pretreated with RuR (30 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. The mitochondrial Ca 2+ concentration ([Ca 2+ ]m) was measured by Rhod-2 AM staining and analyzed by flow cytometry ( A and B ). ( C ) shows the results of confocal microscopy. mPTP opening was determined with calcein AM staining and analyzed by flow cytometry ( D and E ). The levels of cytosolic mtRNA, ND1 and Co-2, ( F and H ) or mRNA of inflammatory signaling molecules ( G and I ) were measured by RT-qPCR. Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A , B , D , and E ). The values are fold changes relative to the mean value of the controls according to the results from RT-qPCR ( F , G , H , and I ). Representative results from at least 3 independent experiments are shown ( C ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( G and I ). Two-way ANOVA with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments ( A , B , D , E , F , and H ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. RIG1, retinoic acid-inducible gene I; ISG15, interferon-stimulated gene 15; IL, interleukin; MDA5, melanoma differentiation-associated protein 5; OAS, 2′-5′-oligoadenylate synthetase, TNF-α, tumor necrosis factor-α

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Concentration Assay, Staining, Flow Cytometry, Confocal Microscopy, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Endoplasmic reticulum calcium release regulates IFN-α-100 and ICs-induced mtRNA release. BMDMs (2 × 10 ) were pretreated with XeC (5 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. The signal and mitochondrial Ca 2+ concentrations ([Ca 2+ ]m) ( A , B , and C ), mPTP opening ( D and E ), cytosolic mtRNA levels ( F and H ), and mRNA levels of inflammatory signaling molecules ( G and I ) were measured as described in Fig. . Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A , B , D , and E ). The values are fold changes relative to the mean value of the controls ( F , G , H , and I ). Representative results from at least 3 independent experiments are shown ( C ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( G and I ). Two-way ANOVA with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments ( A , B , D , E , F , and H ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. RIG1, retinoic acid-inducible gene I; ISG15, interferon-stimulated gene 15; IL, interleukin; MDA5, melanoma differentiation-associated protein 5; OAS, 2′-5′-oligoadenylate synthetase, TNF-α, tumor necrosis factor-α

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Effects of IFN-α-100 or ICs treatment on mitochondrial membrane potential, apoptosis and cell viability. BMDMs (2 × 10 6 ) were treated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. To measure changes in mitochondrial membrane potential, JC-1 green/red staining was performed, after which the cells were analyzed by flow cytometry ( A ). Several approaches to measure cell viability, apoptosis and cell death as described in Materials and Methods were performed. These include the measurements of SYTOX staining ( B ), LDH release ( C ), CCK-8 assays ( D ), annexin-V staining ( E ), and ATP levels ( F ). BMDMs (2 × 10 6 ) were pre-treated with Z-VAD-FMK (20 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. As controls, BMDMs were pre-treated with Z-VAD-FMK (20 µM) for 2 h and then stimulated with TNF-α (20 ng/ml) plus cycloheximide (CHX; 30 µg/ml) for 3 h. The cleavaged products from PARP and caspase 3 were detected by Western blotting ( G ). Cytosolic mtRNA release was measured by RT-qPCR ( H ). Each data point represents one mouse, and the values are fold changes relative to the mean value of the controls ( A and F ). For measuring SYTOX staining and annexin V staining, the values are the percentages of cells with SYTOX Green-positive nuclei (pseudocolored red) or annexin V-positive relative to the total cell population ( B and E ). The values of LDH release are the percentage relative to the mean value of the positive controls ( C ). The values of the CCK-8 and ATP level are the readouts from the absorbance microplate reader and luminescence reader ( D and F ). Representative results from at least 3 independent experiments are shown ( B and G ). One-way ANOVA ( B , D and F ) or two-way ANOVA ( A , C , E and H ) with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Membrane, Staining, Flow Cytometry, CCK-8 Assay, Western Blot, Quantitative RT-PCR, Microplate Reader Absorbance Measurement

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: The sequential events involved in the regulation of mtRNA release into the cytosol by IFN-α-100 and IC treatment. Stimulation of IFN-α-100 and ICs resulted in the release of calcium from the endoplasmic reticulum that entered the mitochondria, induced mitochondrial calcium overload, and triggered the opening of mitochondrial pores, including the oligomerization of voltage dependent anion channel (VDAC) and mPTP. The activation of adenine nucleotide translocase (ANT) also participated in the opening of mitochondrial pores in the inner mitochondrial membrane. Although ANT is not an essential component of the mPTP, ANT can regulate mPTP opening. The opening of the mPTP in the inner mitochondrial membrane and the oligomerization of VDAC1 resulted in the release of mtRNA into the cytosol, which activated its sensors RIG-I and MDA5 as well as several downstream inflammatory signaling pathways to induce innate immunity

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Activation Assay, Membrane, Protein-Protein interactions

Journal: International Journal of Stem Cells

Article Title: Perivascular Stem Cells Suppress Inflammasome Activation during Inflammatory Responses in Macrophages

doi: 10.15283/ijsc19115

Figure Lengend Snippet: The Mechanism underlying the inhibitory effect of PVC-CM on NLRP3 inflammasome via mitochondrial ROS generation. (A) LPS-primed THP-1 cells treated with ATP (2 mM) and the indicated DPI (100 to 10 μ M) for 1 h. Sup and Lys were analyzed by immunoblot using the indicated anti-sera. (B) LPS-primed THP-1 cells were treated with rotenone (20 μ M) and the indicated dosages of PVC-CM for 6 h, after which mitochondrial ROS generation was analyzed by immunoblot. (C) Mitochondrial ROS (MitoSOX) were quantified using a fluorometric microplate reader. (D) Relative fluorescence intensities of Mito Tracker (mitochondria) and MitoSOX (mitochondrial ROS) were analyzed using confocal microscopy. Hoechst was used for nuclear staining. Error bars indicate SD (*p<0.05, **p<0.01, ***p< 0.001).

Article Snippet: The ELISA plates were read-out using a microplate reader (Molecular Devices, CA, USA).

Techniques: Western Blot, Fluorescence, Confocal Microscopy, Staining

Journal: Nature Neuroscience

Article Title: Phosphorylated tau exhibits antimicrobial activity capable of neutralizing herpes simplex virus 1 infectivity in human neurons

doi: 10.1038/s41593-025-02157-0

Figure Lengend Snippet: 3D ReNcell VM cultures were infected with HSV1 to characterize tau’s change in cellular distribution and levels to viral infection. 96 well plates were immunoprobed with anti-p-tau antibodies AT180 and PHF-1 labeled with a fluorescent secondary antibody. Images were collected via confocal microscopy and immunofluorescent representative images show neurons (GFP), HSV1 (RFP), and p-tau (647). Images are representative of experiments repeated in triplicate.

Article Snippet: GFP-expressing ReNcell VM neuronal cell line G10, previously described , was seeded as a 3D culture in a 96-well plate (Greiner Bio-One, 655866) in ReN Differentiation Media (ReN Diff).

Techniques: Infection, Labeling, Confocal Microscopy

Journal:

Article Title: Aging decreases expression and activity of glutathione peroxidase-1 in human endothelial progenitor cells

doi: 10.1016/j.mvr.2009.08.009

Figure Lengend Snippet: Phenotype of EPCs. Mononuclear cells isolated from blood of healthy individuals were cultured on fibronectin-coated plates in EGM-2 for 4 days. The adherent cells (EPCs) were spindle and round shaped (A) (×10 magnification). B: Fluorescent confocal microscopy demonstrated that EPCs were positive for uptake of DiI AcLDL (red) (×60 magnification). C and D: Immunofluorescence confocal microscopy of EPCs (×100 magnification) labeled with Flk-1 (C), and normal mouse IgG (D, as a control). The secondary antibody was conjugated to Texas-Red. Cell nuclei were counterstained with Hoescht 33528.

Article Snippet: After lysis of red blood cells, mononuclear cells were plated at a density of 2×10 7 cells/well on 6-well plates coated with human fibronectin (R & D Systems Inc.) in endothelial growth medium-2 (EGM-2, Cambrex Corp.), which is composed of endothelial cell basal medium-2 (EBM-2), 2% fetal bovine serum, fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor, epidermal growth factor, ascorbic acid, hydrocortisone, and heparin.

Techniques: Isolation, Cell Culture, Confocal Microscopy, Immunofluorescence, Labeling, Control