Journal: Nature Communications

Article Title: Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure

doi: 10.1038/s41467-022-30167-0

Figure Lengend Snippet: a Maximum intensity projection of confocal images of the actin cytoskeleton in prepupal macrophages expressing swip-1 dsRNA marked by eGFP co-expression. Scale bars represent 10 µm. Cells were co-stained with anti-Swip-1 antibody (magenta), DAPI (blue), and phalloidin (white). The arrow marks an eGFP-negative wild-type cell with high Swip-1 expression. b Western blot analysis of lysates of macrophages cells expressing different swip-1 dsRNA transgenes as indicated under the control of the hemolectin -Gal4 driver. The expression of dsRNA transgenes without the Gal4 driver serves as a control. Knock-down efficiency was validated with an anti-Swip-1 specific antibody. Actin served as loading control. c , d Structured illumination microscopic (SIM) images of wild-type prepupal macrophages co-stained for endogenous Swip-1 (magenta), phalloidin (green) and DAPI (blue). Scale bars 10 µm. e Frames of spinning disk microscopic video of a migrating macrophage expressing an Swip-1-eGFP fusion. The arrow marks the localization of Swip-1 in the protruding lamellipodium. Images were taken every 20 s for 30 min (see also Supplementary Movie ). Scale bar 10 µm. f Maximum intensity projection of the confocal image of S2R+ cells transfected with Swip-1-eGFP fusion (magenta), stained with DAPI (blue) and phalloidin (white). Scale bar 10 µm. g Lamellipodia width of S2R+ cells either transfected with cytoplasmic eGFP or Swip-1-eGFP fusion was measured at five regions using Image J and averaged. Boxes indicate 50% (25–75%) and whiskers (5–95%) of all measurements, with black lines depicting the medians. n = eGFP WT: 20, Swip-1-eGFP: 21 cells each from one representative transfection. Two-sided Mann–Whitney test was used, P value: <0.001 (***). All images shown are representative of at least three independent experiments.

Article Snippet: F-actin was visualized using Alexa Fluor-488 or Alexa Fluor-568-conjugated Phalloidin (1:100 dilution, #A12379 #A12380, Invitrogen) and nucleus by DAPI staining (1 µg/mL, #62248, Thermo Scientific).

Techniques: Expressing, Staining, Western Blot, Transfection, MANN-WHITNEY

Journal: Nature Communications

Article Title: Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure

doi: 10.1038/s41467-022-30167-0

Figure Lengend Snippet: a Schematic overview of the swip-1 gene locus. Exons encoding parts of distinct domains are indicated, EF domains in green and coiled-coil region in orange. The target sequence for CRISPR/Cas9 gene modification and generated swip-1 deletions are depicted. b Loss of swip-1 mutants were validated by Western blot analysis. Lysates from ten fly heads of wild-type and different mutant flies were analyzed. Non-specific bands (upper bands) serve as a loading control. All obtained mutants were validated once, hereinafter used swipΔ1 mutant flies were checked regularly in Western blot analysis of head lysates. c , d Confocal images of c wild-type and d swip-1 mutant macrophages were co-stained with phalloidin (white) and DAPI (blue). Scale bars 10 µm. e Quantification of lamellipodia width at the leading edge, n = WT: 64, swip-1 mutant: 61 cells of two independent experiments ( P value: 0.012). f Circularity was measured using Image J shape descriptors, n = WT: 180, mutant: 192 ( P value: >0.001), rescue: 209 cells ( P value: 0.892). g Rescue of swip-1 mutant macrophages re-expressing a Swip-1-eGFP fusion were co-stained with phalloidin (white) and DAPI (blue). The arrow marks mutant cells lacking Swip-1-eGFP that still show an irregular cell shape. h , i Spinning disk microscopy live imaging of randomly migrating macrophages in prepupae with indicated genotypes were tracked by using Imaris 9.3. Track speed mean is color-coded (0.06 to 6 µm/min) ( h ) wild type and ( i ) overexpression of a Swip-1-eGFP (OE) in a wild-type background, j swip-1 mutant, ( k ) rescue with a Swip-1-eGFP, and l rescue with a Swip-1-D82A/D118A-eGFP, respectively. m Quantification of track speed mean, WT: n = 114 tracks, OE: n = 133 tracks ( P value: 0.006), swip-1 mutant: n = 206 tracks ( P value: <0.001), rescue WT: n = 166 tracks ( P values: 0.002 to WT and <0.001 to mutant), rescue SwipD82A/D118A: n = 136 tracks ( P values: 0.594 to WT and <0.001 to mutant). e , f , m Boxes indicate 50% (25–75%) and whiskers (5–95%) of all measurements, with black lines depicting the medians. For statistical analysis, two values indicated by connecting black lines were compared with two-sided Mann–Whitney test, P value: 0.12 (ns), 0.033 (*), 0.002 (**), <0.001 (***). n Frames of spinning disk microscopy video of migrating swip-1 mutant macrophages re-expressing Swip-1-D82A/D118A-eGFP. Images were taken every 20 s for 30 min. A white arrow indicates the direction of movement; yellow arrowheads mark the lamellipodium. Swip-1-D82A/D118A-eGFP localizes to the cell cortex but not to the protruding lamellipodium (see also Supplementary Video ). Scale bar 10 µm. All images shown are representative of at least three independent experiments unless otherwise specified.

Article Snippet: F-actin was visualized using Alexa Fluor-488 or Alexa Fluor-568-conjugated Phalloidin (1:100 dilution, #A12379 #A12380, Invitrogen) and nucleus by DAPI staining (1 µg/mL, #62248, Thermo Scientific).

Techniques: Sequencing, CRISPR, Modification, Generated, Western Blot, Mutagenesis, Staining, Expressing, Microscopy, Imaging, Over Expression, MANN-WHITNEY

Journal: Foods

Article Title: Extracellular Heme Proteins Influence Bovine Myosatellite Cell Proliferation and the Color of Cell-Based Meat

doi: 10.3390/foods8100521

Figure Lengend Snippet: Two-dimensional immunofluorescence stain of isolated bovine muscle satellite cells (BSCs). ( A ) Proliferating bovine satellite stained for DAPI, actin cytoskeleton (Phalloidin), and Pax7, a nuclear marker of satellite cells. Stains show a highly pure satellite cell population, following isolation and pre-plating protocol. ( B ) Following one week of differentiation, cells were stained for DAPI, actin cytoskeleton (Phalloidin), and Troponin T (CT3), a marker of myogenesis. Scale bars are 200 µm.

Article Snippet: For BAMs, secondary antibodies for Troponin T were diluted in blocking solution containing DAPI (Thermo Fisher, #62248, 1:1000) and added to constructs for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Isolation, Marker

Journal: Foods

Article Title: Extracellular Heme Proteins Influence Bovine Myosatellite Cell Proliferation and the Color of Cell-Based Meat

doi: 10.3390/foods8100521

Figure Lengend Snippet: Confocal immunofluorescent imaging of BAMs. BAMs generated from BSC, BSC + Hb, or BSC + Mb (3 mg/mL for both heme proteins) were stained after eight days of differentiation for DAPI, actin cytoskeleton (Phalloidin), and Troponin T (CT3), a marker of myogenesis. Images show multinucleated myotube formation in BSC and BSC + Mb constructs, though not in BSC + Hb constructs. Scale bars are 200 µm.

Article Snippet: For BAMs, secondary antibodies for Troponin T were diluted in blocking solution containing DAPI (Thermo Fisher, #62248, 1:1000) and added to constructs for 1 h at room temperature.

Techniques: Imaging, Generated, Staining, Marker, Construct

Journal: Journal of visualized experiments : JoVE

Article Title: Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

doi: 10.3791/60057

Figure Lengend Snippet: Materials

Article Snippet: Culture medium: Add 50 ng/mL stem cell factor (SCF) and 50 ng/mL thrombopoietin (TPO) to serum-free medium for culture and expansion of hematopoietic cells (see for commercial medium recommended). table ft1 table-wrap mode="anchored" t5 caption a7 Name Company Catalog Number Comments ACK lysing buffer Life Technologies A1049201 B220-biotin BD Bioscience 553086 CD3e-biotin Life Technologies 13-0031-85 CD4-biotin Fischer Scientific BDB553782 CD8-biotin Life Technologies 13-0081-85 CD11 b-biotin BD Bioscience 553309 CD19-biotin BD Bioscience 553784 CD34-FITC eBioscience 11-0341-85 CD48-APC eBioscience 17-0481-82 CD135-biotin eBioscience 13-1351-82 CD150-PerCP/Cy5.5 Biolegend 115922 c-kit-APC/Cy7 Biolegend 105826 Cyclosporin H Millipore Sigma SML1575-1MG DAPI solution (1mg/mL) Life Technologies 62248 Fetal Bovine Serum (FBS) Denville FB5001-H FCCP Millipore Sigma C2920-10MG Grl-biotin Biolegend 108404 IgM-biotin Life Technologies 13-5790-85 Il7Rα-biotin eBioscience 13-1271-85 Nk1.1-biotin Fischer Scientific BDB553163 Phosphate buffered saline (PBS) Life Technologies 10010023 Sca-1-PE/Cy7 eBioscience 25-5981-81 SCF murine PEPROTECH 250-03-10UG StemSpan SFEM medium STEMCELL technologies 9605 Streptavidin-Pacific Blue eBioscience 48-4317-82 Ter119-biotin Fischer Scientific BDB553672 TMRM Millipore Sigma T5428-25MG TPO PEPROTECH 315-14-10UG Verapamil hydrochloride Millipore Sigma V4629-1G Open in a separate window Materials TMRM stock solution: Prepare TMRM (1 μM) solution by dissolving 5 μg of TMRM powder in 10 mL of ethanol.

Techniques: