Journal: Molecular Neurodegeneration
Article Title: An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders
doi: 10.1186/s13024-024-00723-x
Figure Lengend Snippet: Functional phenotype of iMGL derived from the ALSP-CSF1R patient with a c.2350G > A (p.V784M) CSF1R variant. All iMGL were generated using the 2.9 protocol. Quantification ( A ) and images ( B ) of iMGL migratory activity toward ADP assessed by Boyden chamber assay. Cells were concomitantly treated or not with PSB0739. Kruskal–Wallis tests followed by Dunn’s multiple comparison tests were performed. n = 6 healthy control lines and 6 batches of a single ALSP patient line, differentiated side-by-side. White = Hoechst 33342, scale bar = 75 μm in ( B ). Quantification of green fluorescence intensity per cell ( C ) and representative images ( D ) of iMGL exposed to vehicle or pHrodo.™ Green-labelled myelin, opsonized red blood cells (IgG-RBC) or E. coli for three hours and then counterstained with Hoescht 33342. T-tests were performed. n = 3 healthy control lines and 3 batches of a single ALSP patient line, differentiated side-by-side, * p < 0.05. Scale bar = 250 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu m$$\end{document} μ m in ( D ). Quantification of mean fluorescence intensities (MFI; E ) and representative images ( F ) of LAMP1, CD68 and IBA1 immunostaining of iMGL. T-tests were performed. n = 3 healthy control lines and 3 batches of a single ALSP patient line, differentiated side-by-side, ** p < 0.01, *** p < 0.001. Scale bar = 100 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu m$$\end{document} μ m in ( F ). G Cytokine secretion assessed in cell supernatants following a 24-h treatment with vehicle, LPS (100 ng/mL) or Pam 3 CSK 4 (100 ng/mL). A two-way ANOVA was performed, followed by Tukey’s post hoc test. n = 4 healthy control lines and 4 batches of a single ALSP patient line, differentiated side-by-side, *** p < 0.001, ns = non-significant. H qRT-PCR performed after three hours of Pam 3 CSK 4 (100 ng/mL) vs. vehicle treatment. T-tests were performed. n = 3 healthy control lines and 3 batches of a single ALSP patient line, differentiated side-by-side, * p < 0.05
Article Snippet: Cells were incubated at 4 °C overnight with primary antibodies against the following targets: ionized calcium binding adaptor molecule 1 (IBA1; #NC9288364, Fujifilm Wako Chemicals at 1:1000), PU.1 (#2258, Cell Signaling at 1:250), Nanog (#ab21624, Abcam at 1:500), Tra-1–60 (#60064, STEMCell Technologies at 1:200), stage-specific embryonic antigen-4 (SSEA-4; #sc-21704, Santa Cruz Biotechnologies at 1:200), octamer-binding transcription factor 3/4 (OCT3/4; #sc-8628, Santa Cruz Biotechnologies at 1:500), Ki67 (#556003, BD Biosciences at 1:200), lysosomal-associated membrane protein 1 (LAMP1; #9091, Cell Signaling at 1:200) or cluster of differentiation 68 (CD68; #M0814, Dako Omnis at 1:200).
Techniques: Functional Assay, Derivative Assay, Variant Assay, Generated, Activity Assay, Boyden Chamber Assay, Comparison, Fluorescence, Immunostaining, Quantitative RT-PCR
Cell Signaling Technology Inc is a verified supplier 
Cell Signaling Technology Inc manufactures this product 
![Functional phenotype of iMGL derived from the ALSP-CSF1R patient with a c.2350G > A (p.V784M) CSF1R variant. All iMGL were generated using the 2.9 protocol. Quantification ( A ) and images ( B ) of iMGL migratory activity toward ADP assessed by Boyden chamber assay. Cells were concomitantly treated or not with PSB0739. Kruskal–Wallis tests followed by Dunn’s multiple comparison tests were performed. n = 6 healthy control lines and 6 batches of a single ALSP patient line, differentiated side-by-side. White = Hoechst 33342, scale bar = 75 μm in ( B ). Quantification of green fluorescence intensity per cell ( C ) and representative images ( D ) of iMGL exposed to vehicle or pHrodo.™ Green-labelled myelin, opsonized red blood cells (IgG-RBC) or E. coli for three hours and then counterstained with Hoescht 33342. T-tests were performed. n = 3 healthy control lines and 3 batches of a single ALSP patient line, differentiated side-by-side, * p < 0.05. Scale bar = 250 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu m$$\end{document} μ m in ( D ). Quantification of mean fluorescence intensities (MFI; E ) and representative images ( F ) of LAMP1, <t>CD68</t> and IBA1 immunostaining of iMGL. T-tests were performed. n = 3 healthy control lines and 3 batches of a single ALSP patient line, differentiated side-by-side, ** p < 0.01, *** p < 0.001. Scale bar = 100 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu m$$\end{document} μ m in ( F ). G Cytokine secretion assessed in cell supernatants following a 24-h treatment with vehicle, LPS (100 ng/mL) or Pam 3 CSK 4 (100 ng/mL). A two-way ANOVA was performed, followed by Tukey’s post hoc test. n = 4 healthy control lines and 4 batches of a single ALSP patient line, differentiated side-by-side, *** p < 0.001, ns = non-significant. H qRT-PCR performed after three hours of Pam 3 CSK 4 (100 ng/mL) vs. vehicle treatment. T-tests were performed. n = 3 healthy control lines and 3 batches of a single ALSP patient line, differentiated side-by-side, * p < 0.05](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6091/pmc10996091/pmc10996091__13024_2024_723_Fig6_HTML.jpg)