Journal: PLoS ONE

Article Title: Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain

doi: 10.1371/journal.pone.0231597

Figure Lengend Snippet: ICC study to detect the expression of microglia-specific markers (Iba1, A ) and proteins involved in the signaling pathway initiating pain hypersensitivity (P2X4R, B ; and BDNF, C ) in SIM-A9 cell line. SIM-A9 cells (Passage no. 9; P9) were cultured in a 96-well plate in complete growth media for 48 h. The cells were fixed with 4% PFA followed by immunostaining for Iba1, P2X4R, and BDNF. The cells were captured under phase contrast setting (Transmitted) and nuclei were counterstained using Hoechst 33342. The signals for P2X4R were imaged under the green fluorescence channel (excitation 470/22 nm and emission 510/42 nm) whereas Iba1 and BDNF were imaged under red fluorescence channel (excitation 531/40 and emission 593/40). Similar observations were noted in triplicate wells/group in three independent experiments. Scale bar = 100 μm. All images were cropped at the same scale using Adobe Photoshop CC 19.1.9 for clarity and conciseness of the presentation. Full-length images of a representative set ( B ) is presented in .

Article Snippet: We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.

Techniques: Expressing, Cell Culture, Immunostaining, Fluorescence

Journal: PLoS ONE

Article Title: Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain

doi: 10.1371/journal.pone.0231597

Figure Lengend Snippet: Western blot of P2X4R and Iba1 proteins ( A ), and α-tubulin, BDNF and its isoforms in SIM-A9 cell lysates ( B ) collected from different passages. In each blot, the series of bands on the left shows the protein ladder. For P2X4R and Iba1 ( A ), cell lysates collected from P4 and P5, at 30, 40, and 50μg/lane total protein were used to detect P2X4R (green, 43kD) and Iba1 (green, 17kD) on the 800 nm channel. The signals at about 25 kD are due to the non-specific reactivity of secondary antibody. For α-tubulin and BDNF ( B ), cell lysates at 30 and 40 μg/lane total protein were electrophoresed to identify α-tubulin (50 kD), BDNF monomer (14 kD), BDNF dimer (28 kD), and pro-BDNF (37 kD) on the 700 nm and 800 nm channels, respectively. The blots were scanned using an Odyssey imager at intensity setting 5 and processed using ImageStudio 5.2 software. Each sample was run in triplicates ( A ) or duplicates ( B ). C-F . Densitometry analysis of P2X4R, Iba1, BDNF, and BDNF dimer bands was performed using ImageStudio 5.2 software. Signal intensities of P2X4R ( C ) and Iba1 ( D ) bands were normalized for protein loading. Signal intensities of BDNF ( E ) and BDNF dimers ( F ) were first normalized to α-tubulin and then to the amount of protein loaded. Statistical analysis between P4 and P5 was performed by unpaired t-test and Welch’s correction using GraphPad Prism 8.1.2. Asterisks indicate statistically significant differences (* p<0.05). Blots were merged and cropped for the clarity and conciseness of the presentation. Full-length blots are presented in , raw blots A and B .

Article Snippet: We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.

Techniques: Western Blot, Software

Journal: PLoS ONE

Article Title: Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain

doi: 10.1371/journal.pone.0231597

Figure Lengend Snippet: The detection of Iba1, P2X4R, and BDNF expression (green fluorescence, 800 nm channel) and cell nucleus (red fluorescence, 700 nm channel) in 96-well plates using In-cell western (ICW). SIM-A9 cells in 96-well plates were fixed with 4% PFA and were treated using a Li-COR Odyssey blocking solution. Each protein was analyzed in two columns. For each group (nuclear staining, target protein staining, and overlay), the cells in the left columns were immunostained with Iba1, P2X4R, or BDNF primary and secondary antibodies (+/+), whereas the primary antibodies were omitted in the right columns (-/+). All wells were incubated with AF790 secondary antibodies (1:700 dilution) and DRAQ5 (nucleus stain, 1:3000) prior to scanning using a Li-COR Odyssey near-infrared imaging system at intensity setting 5 and plate height 4.0 mm. Three independent experiments were performed with n = 3 wells per group. ICW images of 96-well plates for each protein were merged and cropped for the clarity and conciseness of the presentation. Full-length scans are presented in .

Article Snippet: We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.

Techniques: Expressing, Fluorescence, In-Cell ELISA, Blocking Assay, Staining, Incubation, Imaging

Journal: PLoS ONE

Article Title: Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain

doi: 10.1371/journal.pone.0231597

Figure Lengend Snippet: Effect of LPS ( B, C, and D) and ATP ( E , F , and G ) exposure on the cell viability of SIM-A9 microglia cells (P5 and P6) immediately ( B and E ), 24 h post-treatment ( C and F ), and 48 h post-treatment ( D and G ) using ATP assay. The experimental scheme is shown in A . SIM-A9 cells were cultured in a 96-well-plate at 16,500 cells/well for 48 h. Cells were exposed to 2.5 ng/mL to 50 μg/mL LPS for 2, 4 or 24 h. Cells were treated with 25 nM to 250 μM ATP for 2, 4 or 24 h. The cell viability was evaluated using an ATP assay either immediately, 24 or 48 h post-LPS or ATP treatment. The cell viability of treated cells was calculated relative to control, untreated cells. PEI at 20 μg/mL was used as a positive control. Statistical analysis was performed using GraphPad Prism 8.1.2. Asterisks indicate significant differences (**** p<0.0001, *** p<0.001, ** p<0.005, * p<0.05) compared to the control. The data is representative of two independent experiments and is presented as mean ± standard deviation (SD) of at least n = 4 wells per group.

Article Snippet: We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.

Techniques: ATP Assay, Cell Culture, Positive Control, Standard Deviation

Journal: PLoS ONE

Article Title: Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain

doi: 10.1371/journal.pone.0231597

Figure Lengend Snippet: A ) SIM-A9 cells (P6) were cultured for 48 h and exposed to 1, 50, and 100 μM ATP for 2, 4, 6 and 24 h, then lysed immediately after the treatment for subsequent analysis. B ) For P2X4R and BDNF evaluation, cell lysates were loaded at 50 μg in each lane, and the loading amount for Iba1 and α-tubulin was 30 μg/lane. Human recombinant BDNF protein (14 kD) was loaded at 0.4 μg/lane as a positive control. ATP concentration and time-dependent modulation of the levels of α-tubulin (50 kD, red), P2X4R (43 kD), BDNF (14 kD), BDNF dimer (28 kD) and Iba1 (17 kD) was studied after scanning the blots at 700 nm and 800 nm channels using Odyssey imager at intensity setting 5. C - F . Densitometry analysis of P2X4R, Iba1, BDNF, and BDNF dimer bands was performed using ImageStudio 5.2 software. Normalized signal intensity in Y-axis represents a normalization of protein signal intensity to, first, α-tubulin signal intensity followed by normalization to the signal intensity of control/untreated cells at each time point. Note: C-F demonstrates the quantification of the western blot shown in . It should be noted that the experiment was performed three independent times and the densitometry analysis for representative proteins, BDNF and Iba1, are shown in . Statistical analysis between specified groups was performed using GraphPad Prism 8.1.2. Asterisks indicate statistically significant differences (** p<0.005, * p<0.05). Blots were merged and cropped for the clarity and conciseness of the presentation. Full-length blots are presented in .

Article Snippet: We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.

Techniques: Cell Culture, Recombinant, Positive Control, Concentration Assay, Software, Western Blot

Journal: PLoS ONE

Article Title: Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain

doi: 10.1371/journal.pone.0231597

Figure Lengend Snippet: A ) SIM-A9 microglia cells (P8) were cultured for 48 h and exposed to 50 μM ATP for 2 and 4 h. Cells were fixed with 4% PFA, then treated with blocking solution and Iba1 protein was stained using rabbit Iba1 (1:500) and anti-rabbit AF790 (1:700, green). C ) BDNF was stained using rabbit BDNF (1:500) and anti-rabbit AF790 (1:700, green). Nucleus was stained with DRAQ5 (red). Non-specific binding of secondary antibodies was accounted for by omitting primary antibodies in each group. B and D ) Densitometry analysis of each plate (Iba1 and BDNF) was performed using ImageStudio 5.2 software. Normalized signal intensity in Y-axis represents the normalization of protein signal intensity to nuclear staining followed by subtraction of non-specific signal intensity (-/+, no 1°, only 2° Ab). Statistical analysis between specified groups was performed using GraphPad Prism 8.1.2. Asterisks represent statistically significant differences (* p<0.05). Data are presented as mean ± SD of n = 3 samples. ICW images of 96-well plates for each protein were merged and cropped for the clarity and conciseness of the presentation. Full-length blots are presented in .

Article Snippet: We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.

Techniques: Cell Culture, Blocking Assay, Staining, Binding Assay, Software

Journal: PLoS ONE

Article Title: Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain

doi: 10.1371/journal.pone.0231597

Figure Lengend Snippet: SIM-A9 cells (P8) were cultured for 48 h and incubated with 50 μM ATP for 2 h. Control, untreated cells, and treated cells were fixed with 4% PFA, blocked to prevent non-specific antibody binding, and immunostained with mouse-P2X4R (1:250), rabbit-Iba1 (1:500), and BDNF (1:500) primary antibodies, respectively. P2X4R was counterstained with anti-mouse AF555 (green), Iba1 and BDNF with anti-rabbit AF488 (red), and the nucleus was stained with Hoechst 33342 dye (blue). Images were captured under the phase-contrast setting (Transmitted), DAPI (blue), green or red channels, and merged in the overlay image. Triplicate samples were analyzed in two independent experiments. Scale bar = 100 μm. All images were cropped at the same scale using Adobe Photoshop CC 19.1.9 for clarity and conciseness of the presentation. The full length of a representative phase-contrast image for P2X4R is presented in .

Article Snippet: We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.

Techniques: Cell Culture, Incubation, Binding Assay, Staining

Journal: Nutrients

Article Title: Fatty Acid-Derived N -acylethanolamines Dietary Supplementation Attenuates Neuroinflammation and Cognitive Impairment in LPS Murine Model

doi: 10.3390/nu14183879

Figure Lengend Snippet: Effects of LPS and DS treatment on microglial morphology, proinflammatory cytokines, and NO production. ( a ) Representative images of SIM-A9 microglia cell culture after LPS (1 μg/mL) and DS (0.1, 1 and 10 μg/mL) treatment, scale bar—100 μm. ( b ) Western blot analysis of SIM-A9 microglia cell lysates ( a ) INFγ (25 kDa), TNFα (26 kDa), IL-1β (30 kDa), IL6 (22 kDa), and MHCII (47 kDa) were detected in LPS-activated SIM-A9 cell lysates with β-tubulin protein expression used as a loading control. Original uncropped images of blots are shown in . ( c ) Relative quantity of INFγ, TNFα, IL-1β, IL6, and MHCII in SIM-A9 microglia cell lysates determined with Western blot, n = 4, * p < 0.001—compared to Veh, + p < 0.001—compared to LPS. ( d ) Nitrite production was detected with the Griess test, n = 6 per group. Data are presented as mean ± SEM, +++ p < 0.001 — compared to LPS, *** p < 0.001 — compared to Veh.

Article Snippet: For in vitro studies, we used a microglial cell line SIM-A9 (T0247-GVO-ABM, BioCat, Heidelberg, Germany).

Techniques: Cell Culture, Western Blot, Expressing