Journal: STAR protocols

Article Title: Protocol for measuring interorganelle contact sites in primary cells using a modified proximity ligation assay.

doi: 10.1016/j.xpro.2024.102915

Figure Lengend Snippet: Figure 2. Images of PLA foci in transfected cells (A) Control PLA using IgG mouse antibody and TOMM20 (mitochondria). The PLA image demonstrates the specificity of the assay. (B) PLA for TOMM20 (mitochondria) and calnexin (endoplasmic reticulum). Boxed regions are enlarged in the bottom panels. White arrowheads show non-specific foci. Scale bar: 10 mm for the actual image and 5 mm for zoomed images. (C) Percentage of non-specific PLA foci (Calnexin-TOMM20, CLIMP63-TOMM20) in primary human fibroblast. Each point represents an individual cell. Bars show the average G SD.

Article Snippet: 1 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Mouse IgG isotype control 1:200 Invitrogen 08-6599 Fluorescent-tagged secondary antibody 1:500 Jackson ImmunoResearch 715-605-150; RRID:AB_2340862 (Anti-mouse 647), 711-165-152; RRID:AB_2307443 (Anti-Rabbit cy3) Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium Wisent 319-005-CL Trypsin with EDTA Wisent 325-042-CL Penicillin-Streptomycin Wisent SV30010 Fetal bovine serum Corning 35-077-CV L-glutamine solution Wisent 609-065 EL 1X phosphate-buffered saline, sterile Wisent 311-425-CL Paraformaldehyde Sigma-Aldrich P6148 Bovine serum albumin Sigma-Aldrich A7906 Triton X-100 Sigma-Aldrich 9002-93-1 Epredia Immu-Mount Fisher Scientific 9990412 DAPI (40,6-diamidino-2-phenylindole, dihydrochloride) Thermo Fisher Scientific D1306 Critical commercial assays Duolink in situ green kit mouse/rabbit Sigma-Aldrich DUO92014 Duolink in situ PLA probe anti-rabbit PLUS Sigma-Aldrich DUO92002 Duolink in situ PLA probe anti-mouse MINUS Sigma-Aldrich DUO92004 Duolink in situ wash buffers, fluorescence Sigma-Aldrich DUO82049 Neon Transfection System 10 mL Kit Thermo Fisher Scientific MPK1025 Experimental models: cell lines Primary human fibroblasts (controls and DRP1 mutants) Skin biopsies (Research Ethics Board of the Children’s Hospital of Eastern Ontario (DRP1 mutants)) N/A Recombinant DNA mCherry Addgene 54517 mCherry-tagged CLIMP63 Addgene 136293 Software and algorithms ImageJ National Institutes of Health https://imagej.nih.gov/ij/ R www.r-project.org/ R4.0.5 RStudio http://www.rstudio.com/ Version 2023.03.0 + 386 Other Fisherbrand Cover Glasses Fisher Scientific 1254580 Neon transfection system Thermo Fisher Scientific 10431915 Dry bath Confocal microscope Leica Microsystems Leica SP8

Techniques: Transfection, Control

Journal: bioRxiv

Article Title: Modeling kidney development, disease, and plasticity with clonal expandable nephron progenitor cells and nephron organoids

doi: 10.1101/2023.05.25.542343

Figure Lengend Snippet: (A) Schematic showing the derivation of long-term expandable iNPC lines from hPSCs and its versatile applications. (B–D) Immunofluorescence analyses of 11.3-week human fetal kidney sections for SXI2, p-p38 (B), p-SMAD2/3 (C), and p-SMAD1/5/8 (D). Scale bars, 50 μm. (E and F) Immunofluorescence analyses (E) and quantification (F) of YAP expression in iNPCs cultured in hNPSR-v1 medium supplemented with 0, 2, or 4 µM TRULI for 6 days. Scale bars, 50 μm. (G) Bright-field image of iNPCs cultured in hNPSR-v2 medium for 87 days. Scale bar, 100 μm. (H) Growth curve of iNPCs cultured in hNPSR-v2 in a typical 4-day passage cycle starting from 5,000 cells. (I and J) Immunofluorescence analyses (I) and quantification (J) of iNPCs cultured in hNPSR-v2 medium for 21 days for various NPC marker genes as indicated. Scale bars, 100 μm. (K) Time-course bright-field images showing clonal expansion of iNPCs from one single cell in hNPSR-v2 medium. Scale bars, 100 μm. (L) Immunofluorescence analysis of a single cell iNPC clone for SIX2 and PAX2. Scale bars, 50 μm. (M and N) 3D (M) and 2D (N) PCA plots of bulk RNA-seq data. (O) Heatmap showing gene expression of selected marker genes for undifferentiated NPCs and differentiated kidney cell types, in primary and cultured NPCs, as well as FACS-purified SIX2 + or SIX2 + /PAX2 + iNPCs without further culture (D0-iNPC-SIX2 and D0-iNPC-SIX2/PAX2). Primary SIX2-negative non-NPCs (Pri-SIX2-Neg) isolated from human fetal kidneys were used as negative controls. (P and Q) Bright field (BF) and fluorescence images (P) and quantification (Q) of mCherry expression in iNPCs upon lentiviral overexpression of mCherry (lentiviral OE), or targeted CRISPR/Cas9 knock-in of mCherry-expressing cassette into AAVS1 allele (CRISPR KI). Scale bars, 50 μm. (R and S) Whole-mount immunofluorescence analyses of human nephron organoids generated from iNPCs cultured in hNPSR-v2 medium for 42 days for various nephron marker genes as indicated. Scale bars, 200 μm. (T) Whole-mount immunofluorescence analyses of human nephron organoids generated from iNPCs cultured in hNPSR-v2 for 4 days (upper panels) or 32 days (lower panels) for various nephron marker genes as indicated. Scale bars, 200 μm. (U) Whole-mount immunofluorescence analyses of human nephron organoid generated from iNPCs cultured in hNPSR-v2 medium for 42 days for various nephron marker genes as indicated. Scale bar, 200 μm. Data are presented as mean ± SD. Each column represents counts from three biological replicates (n=3). The significance was determined by two-tailed unpaired Student’s t tests; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. See also Figures S9, S10, and Tables S12 and 13.

Article Snippet: 24 hours later, iNPCs were transfected with a mixture of two plasmids that provide donor DNA for targeted knockin of CAG promoter-driven mCherry expression cassette at the AAVS1 loci (pAAVS1-P-CAG-mCherry, Addgene, # 80492), and that express Cas9 and sgRNA (pXAT2, Addgene, # 80494), at the ratio of 3:1, using Lipofectamine 3000 transfection reagent.

Techniques: Immunofluorescence, Expressing, Cell Culture, Marker, RNA Sequencing Assay, Purification, Isolation, Fluorescence, Over Expression, CRISPR, Knock-In, Generated, Two Tailed Test