Journal: Science (New York, N.Y.)

Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

doi: 10.1126/science.abc3621

Figure Lengend Snippet: (A-B) Number of Ca2+ spikes in MA104-GCaMP cells in RV-infected (SA11cl3-mRuby3) at MOI 0.01 and neighboring (NB)+3 cell, treated with: (A) DMSO, 30μM 18β-glycyrrhetinic acid (18β-gly), or 50μM TAT-Gap19 (Gap19) and imaged ~8–24 hpi or (B) anti-VP7 M60 MAb, anti-NSP4 MAb 622, or rabbit anti-NSP4 antisera 120–147 (Rb Ab) and imaged ~8–24 hpi. (data combined from N=3 independent experiments) (C) Representative images of intercellular calcium waves in MA104-GCaMP cells infected with RV (SA11cl3-mRuby3) at MOI 0.1 and mock- or 10U/mL apyrase-treated (5 U/mL apyrase VI and 5U/mL apyrase VII) and imaged ~10 hpi with (D) representative Ca2+ traces from ~8–25 hpi. (E) Ca2+ spikes in RV-infected and NB cells. (F) Average magnitude of Ca2+ spikes/cell in RV-infected and NB cells, (data combined from N=3 independent experiments). (G) qPCR of purinergic receptor mRNA normalized to 18S mRNA and fold change relative to P2X3 mRNA transcript levels in MA104 cells, (data combined from N=3 independent experiments) (H-I) Ca2+ spikes in MA104-GCaMP cells RV (SA114F)-infected (H) or neighboring (NB) cells (I) and treated with DMSO, 10μM BPTU, 10μM AR-C 118925XX (ARC), 10μM Bx430, or 10μM 5-BDBD (data combined from N=3 independent experiments). (A-B, E-F, H-I) Kruskal-Wallis with Dunn’s multiple comparisons test used. Scale bar = 100 μm. Data represented as mean ± SD, (*p<0.05, ****p<0.0001).

Article Snippet: Chemicals BPTU, AR-C 118925XX, Bx430, 5-BDBD, MRS2179, MRS2279, MRS2500, 10-Panx, suramin, TAT-Gap19, PPADS (Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid), NOC-7, prostaglandin E2, ARL 67156, and ω-agatoxin were purchased from Tocris Bioscience.

Techniques: Infection

Journal: Science (New York, N.Y.)

Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

doi: 10.1126/science.abc3621

Figure Lengend Snippet: (A) J3 jHIE-GCaMP6s monolayers mock- or RV (Ito)-infected produce intercellular calcium waves originating at a cell (arrowheads), imaged at ~4 hpi. (B) IF images of (J3)HIE-GCaMP6s monolayers mock- or RV-infected, fixed at 24 hpi, and immunostained for RV antigen (pink) and counterstained with DAPI (gray). (C) Ca2+ spikes per field-of-view (FOV), in (J3)HIEs mock- or RV-infected and treated with vehicle, 100 μM carbenoxolone (CBX), or 10 μM 10Panx at 8–22 hpi (data combined from N=3 independent experiments). (D) qPCR of purinergic receptor mRNA normalized to 18S mRNA and fold change relative to P2X3 mRNA transcript levels in (J3)HIE monolayers (data combined from N=3 independent experiments). (E) Representative Ca2+ traces/FOV of (J3)HIE-GCaMP6s monolayers either mock- or RV-infected and treated with DMSO, 100 U/mL apyrase, or 10 μM BPTU between 8.5–22 hpi. Mock- or RV-infected (J3)HIEs, treated with DMSO, apyrase, or purinergic receptor blockers. (F) Ca2+ spikes/FOV in (J3)HIE-GCaMP6s monolayers treated with DMSO, 100U/mL apyrase (50 U/mL apyrase VI and 50U/mL apyrase VII), 10 μM BPTU, 10 μM AR-C 118925XX, 10 μM Bx430, or 10 μM 5-BDBD and (G) average magnitude of Ca2+ spikes/FOV for 8.5–22 hpi, (data combined from N=3 independent experiments) (H) Ca2+ spikes/FOV of patient J2 jHIE-GCaMP6s monolayers mock- or RV-infected and treated with DMSO, 10 μM BPTU, 10 μM MRS2179, 10 μM MRS2279, or 10 μM MRS2500 and (I) average magnitude of Ca2+ spikes/FOV for 8–22 hpi, (data combined from N=3 independent experiments). (C,H,I) One-way ANOVA with Bonferroni’s and (F,G) Kruskal-Wallis with Dunn’s multiple corrections test used. Scale bar = 50 μm. Data represented as mean ± SD, (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

Article Snippet: Chemicals BPTU, AR-C 118925XX, Bx430, 5-BDBD, MRS2179, MRS2279, MRS2500, 10-Panx, suramin, TAT-Gap19, PPADS (Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid), NOC-7, prostaglandin E2, ARL 67156, and ω-agatoxin were purchased from Tocris Bioscience.

Techniques: Infection

Journal: Science (New York, N.Y.)

Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

doi: 10.1126/science.abc3621

Figure Lengend Snippet: (A) Rotavirus yield from SA114F-infected MA104 cells treated with DMSO vehicle, 10U/mL apyrase, 10μM BPTU, 10μM AR-C 118925XX, 20μM Bx430, 20μM 5-BDBD, 300μM suramin, or 10μM PPADS by fluorescent focus assay (data combined from N=3 independent experiments). (B) Plaque assay yield of RV (SA114F)-infected MA104-GCaMP (Par) or MA104-GCaMP P2Y1 knockout (KO) cells (data combined from N=3 independent experiments). (C-E) qPCR of mRNA transcripts normalized to 18S mRNA transcripts and fold change relative to the Mock-DMSO transcript levels in (J3)HIE monolayers mock- or RV (Ito)-infected and treated with DMSO vehicle, 100U/mL apyrase, or 10 μM BPTU (data combined from N=3 independent experiments). (F-G) Serotonin secretion from RV (Ito)-infected jHIE (F) monolayers and (G) transwells treated with DMSO, 100U/mL apyrase, 10μM BPTU, or 300nM ω-agatoxin (data combined from N=3 independent experiments). (H) Enteroid swelling assay: 3D jHIE-GCaMP6s enteroids mock- or RV (Ito)-infected and treated with DMSO or 10μM BPTU. Cross-sectional area of the internal lumen (pink outline) used for percent increase between basal and max swelling (left panels). Scale bar = 100μm. (n ≥ 68 HIEs per condition, data combined from N=3 independent experiments) (I-K) C57Bl/6J mouse pups with diarrhea infected with Rhesus RV and vehicle- or BPTU-treated and the (J) mean diarrhea score. (data combined from 4 cages of each condition, 26–30 pups total per condition, mean ± SEM) (K) Summary model of RV-induced ICWs mediated by extracellular ADP. (A-B, F-G) One-way ANOVA with Bonferroni multiple comparisons test, (C-J) Kruskal-Wallis with Dunn’s multiple comparisons, or (I-J) Mann-Whitney tests used. (A-H) Data represented as mean ± SD, (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Chemicals BPTU, AR-C 118925XX, Bx430, 5-BDBD, MRS2179, MRS2279, MRS2500, 10-Panx, suramin, TAT-Gap19, PPADS (Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid), NOC-7, prostaglandin E2, ARL 67156, and ω-agatoxin were purchased from Tocris Bioscience.

Techniques: Infection, Plaque Assay, Knock-Out, MANN-WHITNEY

Journal: eLife

Article Title: CCR1 mediates Müller cell activation and photoreceptor cell death in macular and retinal degeneration

doi: 10.7554/elife.81208

Figure Lengend Snippet: Figure 4. CCR1 is upregulated in a mouse model of photic injury. (A–F) Albino BALB/c mice were exposed to photic injury; 48 hr and 7 days later, retinal sections were prepared and immunostained for Ccr1 (A–C; red) or TUNEL stained (D–F, red); the nuclei were counterstained with DAPI (blue). (G) Real- time quantitative PCR (qPCR) analysis of retinal Ccr1 mRNA measured in control mice and in mice 7 days after photic injury (n=6 mice for each group, Student’s t-test). (H) Ccr1 expression plotted against the b-wave amplitude measured using electroretinography (ERG) in mice 7 days after photic injury.

Article Snippet: Immediately after photic injury was induced, the mice received subcutaneous injections of either the CCR1- specific antagonist BX471 (50 mg/kg body weight; Tocris, Bristol, UK) or vehicle (40% cyclodextrin in saline) every 12 hr for 5 days.

Techniques: TUNEL Assay, Staining, Real-time Polymerase Chain Reaction, Control, Expressing

Journal: eLife

Article Title: CCR1 mediates Müller cell activation and photoreceptor cell death in macular and retinal degeneration

doi: 10.7554/elife.81208

Figure Lengend Snippet: Figure 5. Ccr1 is upregulated in both rd10 mice and senescent mice. (A–F) Retinal sections were prepared from 1-week-old (A, D), 3-week-old (B, E), and 6-week-old (C, F) rd10 mice and immunostained for CCR1; the nuclei were counterstained with DAPI. Note the increased expression of CCR1 (arrows) at 3 weeks (B) and 6 weeks (C), corresponding with reduced ONL thickness (E and F; double-ended arrows). (G–I) Retinal sections were

Article Snippet: Immediately after photic injury was induced, the mice received subcutaneous injections of either the CCR1- specific antagonist BX471 (50 mg/kg body weight; Tocris, Bristol, UK) or vehicle (40% cyclodextrin in saline) every 12 hr for 5 days.

Techniques: Expressing

Journal: eLife

Article Title: CCR1 mediates Müller cell activation and photoreceptor cell death in macular and retinal degeneration

doi: 10.7554/elife.81208

Figure Lengend Snippet: Figure 6. Inhibiting CCR1 reduces the effects of photic injury. (A) Albino BALB/c mice were subjected to photic injury followed by subcutaneous injections of the CCR1 inhibitor BX471 or vehicle for 5 days. Electroretinography (ERG) recordings were then performed, and the amplitude of the b-wave was measured and is plotted against flash intensity (n=12 eyes for each group, Student’s t-test). (B) The number of photoreceptor nuclei was measured at the indicated distances from the optic nerve (n=14 eyes for each group, Student’s t-test). (C–G) Retinal sections were prepared from

Article Snippet: Immediately after photic injury was induced, the mice received subcutaneous injections of either the CCR1- specific antagonist BX471 (50 mg/kg body weight; Tocris, Bristol, UK) or vehicle (40% cyclodextrin in saline) every 12 hr for 5 days.

Techniques:

Journal: eLife

Article Title: CCR1 mediates Müller cell activation and photoreceptor cell death in macular and retinal degeneration

doi: 10.7554/elife.81208

Figure Lengend Snippet: Figure 7. Inhibiting CCR1 modulates the functional properties of M2a hMdɸs. (A–C) M1 (A) and M2a (B) hMdɸs were immunostained for CCR1 (green); magnified views are shown below. (D) Summary of the percentage of CCR1-postive cells measured using cell sorting analysis of CCR1-stained M1 hMdɸ and M2a hMdɸs (n=5 per group, Student’s t-test). (E) Summary of reactive oxygen species (ROS) levels measured in untreated M2a hMdɸs and M2a hMdɸs treated with 0.5 or 5 µM BX471 (n=4 per group, one-way ANOVA with multiple comparisons). (F) Summary of the migration of monocytes treated or not with 10 µM BX471 that migrated toward the M2a hMdɸs using a Boyden chamber (n=3 per group, one-way ANOVA with multiple comparisons). Data shown as mean ± SEM. p-Values indicated by *p<0.05 and **p<0.01. Scale bars: 50 µm (A–C) and 20 µm (insets in A–C).

Article Snippet: Immediately after photic injury was induced, the mice received subcutaneous injections of either the CCR1- specific antagonist BX471 (50 mg/kg body weight; Tocris, Bristol, UK) or vehicle (40% cyclodextrin in saline) every 12 hr for 5 days.

Techniques: Functional Assay, FACS, Staining, Migration

Journal: Autophagy

Article Title: Restoring diabetes-induced autophagic flux arrest in ischemic/reperfused heart by ADIPOR (adiponectin receptor) activation involves both AMPK-dependent and AMPK-independent signaling.

doi: 10.1080/15548627.2017.1358848

Figure Lengend Snippet: Figure 4. Determination of the signaling mechanisms involved in AdipoRon-mediated regulation of cardiomyocyte autophagosome formation in high-fat-induced dia- betic mice. AdipoRon caused significant PRKAA2 phosphorylation (A). Representative western blots (B) and densitometry analysis (C) showing AdipoRon caused significant phosphorylation of ACACA, PIK3C3 (Ser164), BECN1 (Ser93) and BECN1 (Thr119), an effect blocked by compound C (intravenously administered 10 min before AdipoRon), an PRKAA inhibitor. AdipoRon caused significant activation of PtdIns3K, an effect blocked by compound C (D). An in vitro kinase assay demonstrated that activation of PRKAA2 by AMP caused significant BECN1 phosphorylation at Ser93 as well as Thr119 (E: Western blot). AdipoRon promoted BECN1-BCL2L1 dissociation in NC and DB mice (F). DB mice were subjected to sham MI-R or MI-R. Ten min before reperfusion, animals were randomized to receive either vehicle or AdipoRon (intravenous bolus injection). (G) Determination of PRKAA2-induced BECN1 phosphorylation at different sites upon BECN1-BCL2L1 interaction. (A-D) N D 7–8 mice/group; (E-G) representa- tive results from 3–4 repeated experiments. Data were analyzed by one-way ANOVA followed by the Tukey post hoc test for pairwise comparisons. P < 0.05, P < 0.01 between vehicle and AdipoRon-treated MI-R animals; ##P < 0.01 between AdipoRon-treated animals with or without compound C pretreatment (C,D).

Article Snippet: Thirty min after AMPK addition, 1 mg recombinant BCL2L1 (R&D systems, 894-BX) was added and reactions were performed for an additional 30 min at 30 C. Samples were immunoprecipitated with antibody against BECN1, followed by immunoblotting with antibody against BCL2L1.

Techniques: Phospho-proteomics, Western Blot, Activation Assay, In Vitro, Kinase Assay, Injection

Journal: Psychiatry research

Article Title: Genetic variations in evolutionary accelerated regions disrupt cognition in schizophrenia.

doi: 10.1016/j.psychres.2022.114586

Figure Lengend Snippet: Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).

Article Snippet: Chemicals, peptides, and recombinant proteins BAFF Smulski et al., 2017 N/A CD40L Smulski et al., 2017 N/A SARS-Cov2 spike protein Thermofisher Cat# RP-87680 SYK inhibitor (R406) Selleckchem Cat# S2194; CAS 841290-81-1 Dual BCR-ABL/LYN inhibitor (Bafetinib/INNO-406) Selleckchem Cat# S1369; CAS 859212-16-1 PI3Kd inhibitor (nemiralisib/GSK2269557) Selleckchem Cat# S7937; CAS 1254036-71-9 PDK1 inhibitor (BX-795) Selleckchem Cat# S1274; CAS 702675-74-9 FOXO1 inhibitor (AS1842856) Selleckchem Cat# S8222; CAS 836620-48-5 NIK inhibitor (SMI1) Hycultec Cat# HY112433; CAS 1660114-31-7 human monoclonal IgG1l anti-BAFF antibody belimumab (Benlysta) GlaxoSmithKline Pharmaceuticals CAS 356547-88-1 hTACI (aa 31–110)-hIgG1 Fc (aa 245–470) Kowalczyk-Quintas et al., 2019 N/A Fixable viability dye eFluor450 eBioscience Cat# 65-0863-14 Brefeldin A Biolegend Cat# 420601 Critical commercial assays Neon Transfection System 10 mL Kit Thermo Fischer Scientific Cat# MPK1096 NEBuilder HiFi DNA Assembly Cloning Kit New England Biolabs Cat# E5520S jetPEI Polyplus transfection Cat# 101-10N Experimental models: Cell lines DG-75 DSMZ Cat# ACC 83 HEK 293T DSMZ Cat# ACC 635 Oligonucleotides 5ʹ-TCCTCCATGGCAACTACACGTGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD79A.1.AB 5ʹ-AACACCTCGGAGGTCTACCAGGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD79B.1.AA 5ʹ-CCTTCCAAGGACGTCATGCAGGGC-3ʹ Integrated DNA Technologies N/A 5ʹ-TTAATAACATCACCATGCACAGG-3ʹ Integrated DNA Technologies Hs.Cas9.LYN.1.AE 5ʹ-CTCACCGTCCTTGTCTCCGTCGG-3ʹ Integrated DNA Technologies N/A 5ʹ-GTTTGGAAGAGGATCACACTCGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD27.1.AF 5ʹ-CAAATACACGAACTTCTCCC-3ʹ Integrated DNA Technologies Hs.Cas9.TCL1A.1.AE 5ʹ-CTCGGGAAGGTACCAAGGAT-3ʹ Integrated DNA Technologies Hs.Cas9.TNFRSF13B.1.AA Software and algorithms Flow Jo_v10 Flow Jo https://www.flowjo.com/solutions/flowjo RRID: SCR_008520 GraphPad Prism 8.4 GraphPad https://www.graphpad.com/scientific- software/prism RRID: SCR_002798 Image J V.2.3.0 Rasband, 1997-2018 http://imagej.net/ImageJ RRID: SCR_003070 ClustVis Metsalu and Vilo, 2015 https://biit.cs.ut.ee/clustvis/; RRID: SCR_017133 String Szklarczyk et al., 2020 https://www.string-db.org; RRID: SCR_005223 Cell Reports 39, 111019, June 28, 2022 e3

Techniques: Expressing, Western Blot, Control

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: a sAβ42s were added to empty wells (scale bar = 20 μm) or b 12-week-old iPSC neurons at the indicated concentrations. All wells were stained with X04 (blue), Aβ (green), NFL-H (green), and p-Tau S235 (red). Empty wells have Aβ precipitates but no XO4 positive structure. In iPSC neuron wells, there is a dose-dependent increase of X04 staining. A Subset of XO4 is also surrounded by dystrophic neurites (NFL-H and S235 positive axonal swellings). Scale bar = 50 μm. c Microglia treated with sAβ42s ranging from 0 to 5 μM and also treated in combination with IFNγ. The panel below shows a zoomed-in section. Aβ plaques are stained with X04 (blue), microglia are labeled with Actin (green), and IBA1 (red). Scale bar = 50 μm. IFNγ increases plaque formation and plaque interaction. e Quantification of X04 intensity . f Quantification of IBA1 number. d Neuron and astrocytes coculture and triculture of neurons, astrocytes, and microglia were treated with sAβ42s with or without pro-inflammatory cytokine combination (IFNy + IL1b + LPS). Representative images from indicated conditions are shown. The lower panel of zoomed images is shown. Aβ plaque was stained with X04 (blue), dystrophic neurites swellings were stained with NFL-H (green), and microglia were labeled with IBA1 (red). In triple culture, sAβ42s addition led to Aβ plaque formations surrounded by dystrophic neurites and encircled by microglia similar to plaque presentation in vivo. Scale bar = 20 μm. g The area of IBA1 overlap with X04 is quantified. Pro-inflammatory cytokines increased microglia association with plaque. h Microglia increased the X04 plaque area, as measured by the total area of X04 staining. Pro-inflammatory cytokine addition increased the plaque area furthermore. MAP2 areas were also quantified in ( i ). sAβ42s addition caused a severe reduction to neuron culture. Microglia culture provided 25% MAP2 protection from sAβ42s. This protection is lost when pro-inflammatory cytokine is added. Data are presented as mean values +/− SEM and n = 4 wells. Two-way ANOVA with ( e , f , h , i ) Tukey’s or ( g ) Dunnett’s multiple comparisons test.

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: Staining, Labeling, In Vivo

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: a – d Neurons and astrocytes ( a , c ) or neurons, astrocytes, and microglia ( b , d ) were treated with 5 μM sAβ42s and small molecules from a focused screen of known neuroprotective agents at multiple concentrations (50, 25, 12.5, and 6.25 μM (double culture), 50, 12.5, 3.1, and 0.78 μM (triple culture). Results were graphed as Synapse % rescue versus MAP2 % rescue ( a , b ) and Beta III tubulin % rescue versus MAP2 % rescue ( c , d ). Small molecules that prevented toxicity in dendrites (MAP2), synapses (Synapsin 1/2), cell count (CUX2), or axons (NFL-H) at or above 30% were considered hits (red dotted line); anti-Aβ antibody used as a positive control that prevented all types of toxicity. e – g Further validation of top hits DLKi ( e ), Indirubin-3′-monoxime ( f ), and AZD0530 ( g ) from the focused screen by IC50 curves against MAP2 (blue), Synapsin 1/2 (green), CUX2 (red), and NFL-H (purple). h sAβ42s treatment induced expression of p-cJun (green) in the nucleus (HuCD, red). i Quantification of MAP2 (blue), HuC/D (red), p-cJun (green) staining indicated an increase in cJun phosphorylation with prolonged sAβ42s treatment. j 22-week-old iPSC neuron culture treated with sAβ42s showed dose-dependent, sustained phosphorylation of cJun as shown by western blot. GAPDH served as the loading control. k Western blot quantification of p-cJun induction normalized to GAPDH. l – o Inhibition of known components of DLK-JNK-cJun pathway using small molecules VX-680 ( l ), GNE-495 ( m ), PF06260933 ( n ), JNK-IN-8 ( o ) prevents sAβ42s-induced neural toxicity in all measured markers in a dose-dependent manner. Data are presented as mean values +/− SEM and n = 4 wells ( e – g , I , k – o ). IC50 curves fitted by Prism software ( e – g , m – o ).

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: Cell Counting, Positive Control, Biomarker Discovery, Expressing, Staining, Phospho-proteomics, Western Blot, Control, Inhibition, Software

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: a Schematic showing sAβ42s labeled by HiLyte-555 and pHrodo Green continuously fluoresce red, but only fluoresce green under intracellular pH 5 conditions. b Quantitative analysis of red Aβ plaque area and green internalized Aβ. Internalized green Aβ outpace the red extracellular Aβ plaque formation, indicating active Aβuptake throughout the 7 days and proceed before the appearance of red Aβ plaques. c Example images of the plaque formation time-lapse movie. Four different plaque formations are retrospectively labeled. sAβ42s are first internalized by microglia (green) before plaque formation (red) in the center of the cultured microglia. Scale bar = 100 μm. d iPSC-derived microglia were treated with 5 μM sAβ42s, then fixed and stained after 30 min, 6 h, 1 day, and 4 days. Microglia (IBA1, red) internalize small Aβ puncta (green; white—the second row) indicated by white arrowheads (green) after 30 min, then externalize these puncta as large aggregates that are faintly X04 positive (blue; white—bottom row) indicated by white arrows, then form large, extracellular X04-positive plaque structures surrounded by microglia from 1 to 6 days. e Human iPSC-derived microglia were treated with 5 μM soluble Aβ species and various dynamin inhibitors (Dynasore, Dynole 4a, Dynole 34-2) at 0.6 μM for 24 h and plaque-like structures (Methoxy-X04-positive) were quantified as a percentage of control. Treating with dynamin inhibitors decreased plaque formation approximately fourfold in all conditions. f A summary of steps of microglia plaque formation observed. Data are presented as mean values +/− SEM and n = 4 wells. One-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: Labeling, Cell Culture, Derivative Assay, Staining, Control

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: a , b iPSC neurons and astrocytes treated with 5 μM sAβ42s followed by serial dilutions of anti-gD and anti-Aβ antibodies with IgG1 and LALAPG backbones with and without iPSC microglia. Results for dendrite protection (MAP2 area) ( a ) and synapse protection (synapsin count) ( b ) were analyzed via IC50 curve fitting using Prism software. Microglia provide baseline protection as shown by an upward shift in the anti-gD graph when microglia are added (gD IgG1 alone, blue; gD IgG1 + microglia, dark blue). Anti-Aβ antibody backbones protect dendrites and synapses similarly without microglia (Anti-Aβ IgG1, red; Anti-Aβ LALAPG, green) and with microglia (Anti-Aβ IgG1, dark red; Anti-Aβ LALAPG, dark green). c , d Neuron, astrocyte, microglia triculture treated with 5 μM sAβ42s (solid lines) and pro-inflammatory cytokines (dashed lines), then serial dilutions of gD antibody (black lines) and anti-Aβ antibody (red lines) were added. c Basal dendrite protection (MAP2 area) is lost in a neuroinflammatory environment, and anti-Aβ shows dose-dependent efficacy. d Plaque formation (Methoxy-X04 total intensity) increases in pro-inflammatory conditions, however, anti-Aβ shows similar plaque reduction. e iPSC microglia (red) were treated with 5 sAβ42s and serial dilutions of anti-Aβ antibody; no cells wells were used as control (blue). The supernatant was collected and anti-Aβ ELISA was performed to measure total Aβ concentration. Anti-Aβ antibody treatment increases soluble Aβ species present in the culture supernatant. f Summary of sequential events in the iPSC AD model. Data are presented as mean values +/− SEM and n = 4 wells. Two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: Software, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: ApoE genotype status.

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: