Journal: Scientific Reports

Article Title: The BET inhibitor attenuates the inflammatory response and cell migration in human microglial HMC3 cell line

doi: 10.1038/s41598-021-87828-1

Figure Lengend Snippet: Effects of LPS and JQ1 on HMC3 cells and the expression of inflammatory response-related genes in LPS- and JQ1-treated cells. ( A ) Morphology of HMC3 cells after treatment with LPS, JQ1, and LPS + JQ1 for 4 h. ( B ) HMC3 cells were cultured for 4 h after treatment with LPS (100 ng/ml) and JQ1 (500 nM) and stained for IBA1 (green fluorescence), a microglia-lineage marker. Nuclei were counterstained with DAPI (blue). ( C ) HMC3 cells were treated with LPS and JQ1 at different concentrations for different durations (4 h, 24 h, and 48 h). The viability of the HMC3 cells was determined using the WST-1 assay. ( D ) HMC3 cells were treated with LPS at different doses (10, 100, and 1000 ng/ml) for 4 h. Inflammatory genes were significantly upregulated in cells treated with LPS compared to DMSO-treated control cells (upper panel). HMC3 cells were treated with LPS (100 ng/ml) and JQ1 at different doses (50, 500, and 5000 nM) for 4 h. Inflammatory genes were significantly downregulated in cells treated with JQ1 (bottom panel). Gene expression was normalized to GAPDH transcript levels. The data represent three independent experiments. The values are the mean ± SEM of triplicate experiments (* p < 0.05 and ** p < 0.001).

Article Snippet: HMC3 human microglial cells were purchased from the Korean Cell Line Bank (Seoul, Korea).

Techniques: Expressing, Cell Culture, Staining, Fluorescence, Marker, WST-1 Assay, Control, Gene Expression

Journal: Scientific Reports

Article Title: The BET inhibitor attenuates the inflammatory response and cell migration in human microglial HMC3 cell line

doi: 10.1038/s41598-021-87828-1

Figure Lengend Snippet: Differentially expressed genes in LPS- and LPS + JQ1-treated HMC3 cells. ( A ) Heat map showing the top 50 upregulated genes in HMC3 cells treated with LPS and LPS + JQ1 for 4 h determined by RNA-seq ( p ≤ 0.05 and fold change ≥ 1.5 log2). Each experiment was performed in experimental triplicates (n = 3) for each condition, and the results were individually combined. The color scale shown in the heat map represents the log 2 fold change values. Red cells indicate upregulated gene, while blue cells indicate downregulated genes. The p-value with an asterisk attached in the cell represents * p < 0.05, ** p < 0.01, and *** p < 0.001. ( B ) GO term analysis of the biological processes associated with up- and downregulated genes after LPS (100 ng/ml) and JQ1 (500 nM) treatment for 4 h. GO analysis of the number of genes shown in the van diagram. ( C ) Biological pathway analysis of DEGs in cells treated with LPS and LPS + JQ1 using IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis ) showed up- and downregulated genes.

Article Snippet: HMC3 human microglial cells were purchased from the Korean Cell Line Bank (Seoul, Korea).

Techniques: RNA Sequencing

Journal: Scientific Reports

Article Title: The BET inhibitor attenuates the inflammatory response and cell migration in human microglial HMC3 cell line

doi: 10.1038/s41598-021-87828-1

Figure Lengend Snippet: JQ1 inhibits a specific subset of LPS-inducible inflammatory genes. ( A ) Heat map showing chemokine/cytokine and interferon response gene expression in HMC3 cells treated with LPS (100 ng/ml) and JQ1 (500 nM) for 4 h. The color scale shown in the heat map represents the log 2 fold change values. ( B ) IPA downstream effects analysis presenting the mapping of most significant genes related to inflammatory response genes in LPS-treated cells. The DEGs are colored by predicted activation state, such as activated (red) or suppressed (green). The edges connecting the nodes are colored orange (leading to activation of the downstream node), blue (leading to inhibition), and yellow (if the findings underlying the relationship are inconsistent with the state of the downstream node). ( C ) Expression levels of inflammation-related genes were analyzed by qRT-PCR and normalized to GAPDH transcript levels. Overall, LPS-induced inflammatory genes were inhibited by JQ1. The data represent three independent experiments. The values are the mean ± SEM of triplicate experiments (** p < 0.001).

Article Snippet: HMC3 human microglial cells were purchased from the Korean Cell Line Bank (Seoul, Korea).

Techniques: Gene Expression, Activation Assay, Inhibition, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: The BET inhibitor attenuates the inflammatory response and cell migration in human microglial HMC3 cell line

doi: 10.1038/s41598-021-87828-1

Figure Lengend Snippet: IRF1 regulates migration-related genes in LPS- and LPS + JQ1-treated HMC3 cells. ( A ) IPA upstream regulator analysis of DEGs in LPS-, JQ1- and LPS + JQ1-treated HMC3 cells. The color scale shown in the heat map represents the activation z-score value. ( B ) TF-binding motif analysis to determine TF-binding site enrichment in the promoters of migratory genes. Putative binding sites for IRF1was significantly enriched in LPS-induced migratory genes. ( C ) Expression of RELA and IRF family genes were analyzed by qRT-PCR and normalized to GAPDH transcript levels. Among these IRF family genes, IRF1 and IRF2 were significantly upregulated in LPS-treated cells. The data represent three independent experiments. The values are the mean ± SEM of triplicate experiments (** p < 0.001). ( D ) IPA upstream regulator analysis identified key regulatory gene IRF1, and it targets genes in LPS-treated cells. Of the genes regulated by IRF1, only migration-related genes are shown in bold and in red. The migration-related genes were highly correlated with IRF1. ( E ) Log 2 fold changes and p -value in the expression of IRF1 target genes. ( F ) IPA network analysis of genes related to the inflammatory response and migration in LPS-treated cells. Of the genes identified, only chemotaxis-related genes are shown in bold and in red. Most inflammation-related genes overlap with migration-related genes.

Article Snippet: HMC3 human microglial cells were purchased from the Korean Cell Line Bank (Seoul, Korea).

Techniques: Migration, Activation Assay, Binding Assay, Expressing, Quantitative RT-PCR, Chemotaxis Assay

Journal: Journal of Neuroinflammation

Article Title: The human microglial HMC3 cell line: where do we stand? A systematic literature review

doi: 10.1186/s12974-018-1288-0

Figure Lengend Snippet: Historical reconstruction of the distribution process of the human microglial clone 3 cell line. The human microglial clone 3 cell line was developed in the laboratory of Prof. M Tardieu, Paris, in 1995 (red circle). As shown in the picture, clone 3 has been distributed worldwide, with the acronym of CHME3 cells (blue boxes) or HMC3 cells (green boxes). Distribution followed two main pathways, either directly from Prof. Tardieu’s laboratory (black thick arrows) or indirectly by the first recipient laboratory (black dotted arrows). A second main distributor of the CHME3 cell line is the laboratory of Prof. A Basu, National Brain Research Centre (NBRC), India (purple circle). Since 2014, this laboratory appears to be the main distributor of the CHME3 cells in India. However, we could not trace on the timeline when the cell line was transferred from the laboratory of Prof. Tardieu to NBRC. In addition, we identified several studies (not reported in the schematic), in which the CHME3 cells were used without any indication of the source, and one study in which the cell line was provided by an Academic institution without any link to published data. In 2016, the HMC3 cells were transferred to ATCC®, USA (orange box) and authenticated and distributed under the catalog designation of HMC3 (ATCC®CRL-3304)

Article Snippet: To the best of our knowledge, two cell lines are commercially available, the human microglial clone 3 cell line, HMC3 [ , – ] and the “Immortalized Human Microglia - SV40”, developed and distributed by Applied Biological Materials (Vancouver, Canada) [ – ].

Techniques:

Journal: Journal of Neuroinflammation

Article Title: The human microglial HMC3 cell line: where do we stand? A systematic literature review

doi: 10.1186/s12974-018-1288-0

Figure Lengend Snippet: Antigenic profile of the human microglial clone 3 cell line

Article Snippet: To the best of our knowledge, two cell lines are commercially available, the human microglial clone 3 cell line, HMC3 [ , – ] and the “Immortalized Human Microglia - SV40”, developed and distributed by Applied Biological Materials (Vancouver, Canada) [ – ].

Techniques: Western Blot

Journal: Journal of Neuroinflammation

Article Title: The human microglial HMC3 cell line: where do we stand? A systematic literature review

doi: 10.1186/s12974-018-1288-0

Figure Lengend Snippet: Production of cytokines, chemokines, and other inflammatory mediators

Article Snippet: To the best of our knowledge, two cell lines are commercially available, the human microglial clone 3 cell line, HMC3 [ , – ] and the “Immortalized Human Microglia - SV40”, developed and distributed by Applied Biological Materials (Vancouver, Canada) [ – ].

Techniques:

Journal: Journal of Neuroinflammation

Article Title: The human microglial HMC3 cell line: where do we stand? A systematic literature review

doi: 10.1186/s12974-018-1288-0

Figure Lengend Snippet: HMC3 cell morphology and labeling of cytoskeletal F-actin filaments. a – b The human microglial cell line HMC3 as it was observed by phase-contrast microscopy at in vitro day 1 ( a ), and when cells reached the confluency ( b ). × 10 magnification, scale bar 100 μM. c – e A representative example of confocal images (1024 × 1024 pixels) acquired at × 20 magnification with a confocal laser scanning system (A1+, Nikon). Cells were grown on glass coverslips for 24 h, and their morphology was evaluated by labeling the cytoskeletal F-actin filaments with tetramethylrhodamine (TRITC)-conjugated phalloidin (red fluorescence). Cells were counterstained with the nuclear probe, 4′,6-damidino-2-phenylindole dihydrochloride (DAPI, blue fluorescence). The merged image is shown in ( e ). Scale bar 50 μM

Article Snippet: To the best of our knowledge, two cell lines are commercially available, the human microglial clone 3 cell line, HMC3 [ , – ] and the “Immortalized Human Microglia - SV40”, developed and distributed by Applied Biological Materials (Vancouver, Canada) [ – ].

Techniques: Labeling, Microscopy, In Vitro, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: The human microglial HMC3 cell line: where do we stand? A systematic literature review

doi: 10.1186/s12974-018-1288-0

Figure Lengend Snippet: HMC3 cell expression of microglial lineage markers. a – f Representative example of confocal images (1024 × 1024 pixels) acquired at × 20 magnification with a confocal laser scanning system (A1+, Nikon). HMC-3 cells immuno-labeled for IBA1 (green fluorescence, d , f ) and DAPI stained (blue fluorescence, a , b ) are shown. Merged images are shown in ( e , f ). Control experiments performed by omitting the primary antibody are shown in ( a , c, e ). No green fluorescence was present ( c , e ), indicating neither spontaneous fluorescence nor non-specificity of the secondary antibody. Scale bar 50 μM. g – h Total RNA was prepared from human microglial HMC3 cells 24 h after incubation in complete growth medium and retrotranscribed using random hexamers. Real time (Q)-PCR analysis for the mRNA levels of IBA1, CX3CR1, CCR2, P2RY12, TMEM119, and CSF1-R was carried out according to our standard protocols . g , h panels show a representative gel image of PCR products obtained at the end of the analysis from two different RNA samples for each condition. In the last line is shown the actin amplification, as positive control

Article Snippet: To the best of our knowledge, two cell lines are commercially available, the human microglial clone 3 cell line, HMC3 [ , – ] and the “Immortalized Human Microglia - SV40”, developed and distributed by Applied Biological Materials (Vancouver, Canada) [ – ].

Techniques: Expressing, Labeling, Fluorescence, Staining, Control, Incubation, Amplification, Positive Control

Journal: Journal of Neuroinflammation

Article Title: The human microglial HMC3 cell line: where do we stand? A systematic literature review

doi: 10.1186/s12974-018-1288-0

Figure Lengend Snippet: Phenotypical characterization of HMC3 cells under basal conditions and in response to IFNγ. HMC3 cells were plated at the density of 30,000 cells/cm 2 in T25 flasks, and grown for 3 days when cells were almost confluent. In the experiments in which INFγ was used, cells were stimulated for the last 36 h. Controls did not receive any stimulus for the same time period (“resting” HMC3 cells). For detection of intracellular antigens, aliquots of 1 × 10 6 cells in 100 μl were fixed using BD cytofix (BD Pharmingen) at 4 °C for 30 min, and permeabilized with BD FACS permeabilizing solution at 4 °C for 30 min. Cells were then stained using the following antibodies: PE-conjugated anti Human GFAP mouse Mab, BD Bioscience ( a ), and PE-CF594-conjugated anti Human CD68 mouse Mab, BD Bioscience ( e ), according to the manufacturer’s instructions. For the evaluation of surface antigens, aliquots of 5 × 10 5 cells in 100 μl were directly incubated in PBS buffer containing the following antibodies: PE-CF594-conjugated anti Human HLA-DR mouse Mab, BD Bioscience ( b ); FITC-conjugated anti Human CD14 mouse Mab, BD Pharmingen ( c ); and PE-conjugated anti Human CD11b mouse Mab, E-Bioscience ( d ). Cells were analyzed by the 6-parameter (2 scatter and 4 fluorescence signals) Coulter Epics XL flow cytometer (Beckman-Coulter). Control histograms (white histograms) indicate level of cell autofluorescence in the emission wavelength that pertains the fluorochrome-conjugated Mab. Panel ( a ) shows expression of GFAP on HMC3 cells at passage 4 (dark gray), and on the human glioblastoma U373 cell line (light gray) that constitutively expresses GFAP. As autofluorescence signal in the two cell lines was similar, only one representative histogram is plotted in panel ( a ) (white histogram). As shown in this panel, the HMC3 cells stain negatively for GFAP (the dark gray histogram completely overlaps the background histogram), whereas the U373 cells express the target antigen. Two different populations expressing GFAP at different levels were identified (light gray plot): 68% of the U373 cells express GFAP at low level (GFAP-dim), whereas 32% of the cells express GFAP at higher level (GFAP-high). Panels ( b – e ) show results from HMC3 cells obtained at passage 7. In these panels, white histograms indicate cell autofluorescence, light gray histograms and dark gray histograms are representative of control (“resting”) HMC3 cells and IFNγ-treated HMC3 cells, respectively. Gating strategy: all analyses were obtained after gating according to morphological characteristics (not shown). As levels of autofluorescence did not change according to cell treatment, one representative example is plotted for each emission wavelength. Linear regions are used for calculating percentage of positive cells (plots A–B). Central tendency of CD68 expression on HMC3 cells in the different experimental conditions is represented by the mean fluorescence intensity (MFI, see text)

Article Snippet: To the best of our knowledge, two cell lines are commercially available, the human microglial clone 3 cell line, HMC3 [ , – ] and the “Immortalized Human Microglia - SV40”, developed and distributed by Applied Biological Materials (Vancouver, Canada) [ – ].

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

Journal: Journal of Neuroinflammation

Article Title: The human microglial HMC3 cell line: where do we stand? A systematic literature review

doi: 10.1186/s12974-018-1288-0

Figure Lengend Snippet: Expression of the human class I MHC antigens on HMC3 cells. a HMC3 were plated at the density 30,000 cells/cm 2 in T25 flasks, and grown for 3 days when cells were almost confluent. For the detection of class I MHC surface antigens, aliquots of 5 × 10 5 cells in 100 μl were directly incubated with FITC-conjugated anti human HLA-ABC mouse Mab, BD Bioscience. This antibody is specific for human MHCI antigens, and does not cross react with rat MHCI antigens. Cells were analyzed by the 6-parameter (2 scatter and 4 fluorescence signals) Coulter Epics XL flow cytometer (Beckman-Coulter). Results from HMC3 cells at passage 8 are shown. Gating strategy: the analysis was obtained after gating according to morphological characteristics (not shown). Background histogram (white) indicates level of cell autofluorescence. A linear region was used for calculating percentage of positive cells. b Gel image of PCR products obtained at the end of a HLA Locus B specific amplification protocol. The 922 bp amplicons were separated by electrophoresis through 1.5% agarose gels containing 0.1 μg/ml ethidium bromide. Lines 2–3 amplification products from genomic DNA extracted by HMC3 (ATCC®CRL-3304) cells; lines 4–5 amplification products from a positive control, i.e., an anonymous human genomic DNA sample provided by ViiV Healthcare Ldt

Article Snippet: To the best of our knowledge, two cell lines are commercially available, the human microglial clone 3 cell line, HMC3 [ , – ] and the “Immortalized Human Microglia - SV40”, developed and distributed by Applied Biological Materials (Vancouver, Canada) [ – ].

Techniques: Expressing, Incubation, Fluorescence, Flow Cytometry, Amplification, Electrophoresis, Positive Control

Journal: Journal of Neuroinflammation

Article Title: The human microglial HMC3 cell line: where do we stand? A systematic literature review

doi: 10.1186/s12974-018-1288-0

Figure Lengend Snippet: Gene expression analysis of the HMC3 (ATCC®CRL-3304) cells

Article Snippet: To the best of our knowledge, two cell lines are commercially available, the human microglial clone 3 cell line, HMC3 [ , – ] and the “Immortalized Human Microglia - SV40”, developed and distributed by Applied Biological Materials (Vancouver, Canada) [ – ].

Techniques: Gene Expression, Control

Journal: Chemical Science

Article Title: Monitoring phagocytic uptake of amyloid β into glial cell lysosomes in real time †

doi: 10.1039/d1sc03486c

Figure Lengend Snippet: Synthesis and characterization of Aβ pH . (A) The Aβ pH is synthesized by conjugating the amine-reactive pH-sensitive Protonex Green dye to the side chain amine groups of the lysine residues and the N-terminal of human Aβ 1–42 peptide. (B) The pH-sensitivity of the Aβ pH probe characterized at different concentrations from 0.1 μM to 5.0 μM. Increased fluorescence is observed at acidic pH values of ∼5.0 to ∼2.0, covering the pH range of the intracellular acidic organelles. (C) Atomic force microscopy topographic images of Aβ pH oligomers compared to synthetic Aβ oligomers. Left-2D topographic image of Aβ pH and synthetic Aβ oligomers. Right-3D image (2 × 2 μm x – y ). (D) Live cell imaging of the phagocytic uptake of 1 μM Aβ pH by BV2 and N9 mouse microglia and by HMC3 human microglia over 24 hours. (E) Quantification of Aβ pH phagocytic score by BV2, N9, and HMC3 microglial cells from the live cell images. (F) The phagocytic uptake of Aβ pH by BV2 cells is measured and quantified via flow cytometry analysis. Dot plot shows live (PI − ) and Aβ pH+ cells. No green fluorescence is measured in unstained cells (UC) and in dead cells stained with the PI only whereas green fluorescence is measured in cells treated with 0.5 and 5.0 μM Aβ pH for 1 hour (higher fluorescence is seen in cells exposed to the higher concentration of Aβ pH ). Data shown in terms of % max, by scaling each curve to mode = 100% ( y -axis).

Article Snippet: HMC3 human microglial cell line was a gift from Dr Jianming Li (Purdue University, USA) who originally obtained the cells from ATCC.

Techniques: Synthesized, Fluorescence, Microscopy, Live Cell Imaging, Flow Cytometry, Staining, Concentration Assay

Journal: Chemical Science

Article Title: Monitoring phagocytic uptake of amyloid β into glial cell lysosomes in real time †

doi: 10.1039/d1sc03486c

Figure Lengend Snippet: Fluorescence of internalized Aβ pH is retained in fixed cells. (A) Confocal images of fixed HMC3, N9, and BV2 cells showing the uptake of Aβ pH (green). Cells are stained for acidic intracellular organelles (LysoTracker Red, confirming co-localization of the Aβ pH within the acidic intracellular organelles) and nuclei (DAPI, blue). No antibody is required to detect Aβ pH . (B) Primary mouse microglia grown in defined, reduced-serum media phagocytose Aβ pH ex vivo . Cells are fixed and stained for nuclei and show Aβ pH colocalized in the acidic organelles with LysoTracker Red. (C) The phagocytic uptake of Aβ pH by primary microglia is measured and quantified via flow cytometry analysis. Dot plot shows live (ZV − ) and Aβ pH+ cells. No green fluorescence is measured in unstained cells (UC) or dead cells stained with the ZV live/dead stain only whereas green fluorescence is measured in cells treated with 0.5, 1.0, and 2.0 μM Aβ pH for 1 hour. Data shown in terms of % max, by scaling each curve to mode = 100% ( y -axis). (D) Primary immunopanned rat astrocytes also phagocytose Aβ pH in serum-free conditions. Cells are fixed and stained for astrocyte specific GFAP antibody (red) and nuclei. (E) Uptake of Aβ pH over time by primary immunopanned astrocytes as observed in live cells in real time. (F) Quantification of uptake of 0.5, 1.0, and 2.0 μM Aβ pH by primary astrocytes. Data are mean ± SEM, n = 8 separate wells per group/timepoint.

Article Snippet: HMC3 human microglial cell line was a gift from Dr Jianming Li (Purdue University, USA) who originally obtained the cells from ATCC.

Techniques: Fluorescence, Staining, Ex Vivo, Flow Cytometry