Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-10-dependent S100A8 gene induction in monocytes/macrophages by double-stranded RNA.

doi: 10.4049/jimmunol.0802683

Figure Lengend Snippet: FIGURE 3. Poly(I:C) induces S100 mRNA in human monocytes and macrophages and IL-10 is involved. A, Monocytes were untreated or stimulated with LPS (500 ng/ml) or poly(I:C) (50 g/ml); means SD of duplicate measurements of cells from five donors. , p 0.05, compared with relative S100:-actin mRNA ratios of control. B, Monocytes were untreated or stimulated with poly(I:C) (50 g/ml) with or without IL-10 (2 or 10 ng/ml). Data represent S100 mRNA relative to poly(I:C)-stimulated samples without IL-10; means SD of three separate experiments. , p 0.05, compared with relative S100:-actin mRNA ratios of poly(I:C) alone. Human macrophages (C and D) or monocytes (E and F) were untreated or stimulated with LPS (500 ng/ml) or poly(I:C) (50 g/ml) with or without anti-IL-10 mAb (5 g/ml) or IgG isotype control for 24 h, or pretreated with 2-AP (4 mM) for 4 h. S100A8 (C and E) and S100A9 (D and F) mRNA levels relative to -actin mRNA were quantitated. Data represent means SD of duplicate measurements and are representative of two to three independent experiments. IL-10 mRNA levels relative to -actin mRNA were also quantitated. Ctr, Control.

Article Snippet: Affinity-purified rabbit anti-murine IL-10, anti-human IL-10 mAb, and mouse IgG2b isotope control were from R&D Systems.

Techniques: Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-10-dependent S100A8 gene induction in monocytes/macrophages by double-stranded RNA.

doi: 10.4049/jimmunol.0802683

Figure Lengend Snippet: FIGURE 7. S100A8 expression in lung tissues from mice infected with influenza A virus and human alveolar A549 epithelial cells. A–E, Lungs were harvested at the day of infection (A) or 5 days (B); arrows indicate pos- itive neutrophil staining) 6 days (C), 8 days (D), or 12 days (E) postinfec- tion. Samples were stained with anti- murine S100A8 IgG. Immunostaining in tissues treated with a negative con- trol IgG were similar to samples shown in A (data not shown). Original magnification, 1000. F, Human A549 epithelial cells were incubated with 2.5, 10, 25, or 50 g/ml poly(I: C). Controls were untreated (Ctr) or treated with 20 ng/ml LPS. Cells were harvested 24 h poststimulation and S100A8 mRNA was quantitated by real-time RT-PCR; -actin mRNA was endogenous control. Data are means SD of duplicate measure- ments and are representative of three independent experiments.

Article Snippet: Affinity-purified rabbit anti-murine IL-10, anti-human IL-10 mAb, and mouse IgG2b isotope control were from R&D Systems.

Techniques: Expressing, Infection, Virus, Staining, Immunostaining, Incubation, Quantitative RT-PCR, Control

Journal: Nature protocols

Article Title: Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

doi: 10.1038/s41596-022-00681-y

Figure Lengend Snippet: a-e, See Table 1 for further descriptions. Representative IHC images showing key checkpoints throughout the differentiation procedure of WA25 hESC-derived skin organoids. a, Day 18 image representing the ECAD+TFAP2A+ epithelium and TFAP2A+ CNC or mesenchymal cells surrounding the aggregate; the presence of these cell populations should be checked between days 6–20 of differentiation. b, Day 55 images showing TFAP2A+KRT5+ and KRT17+KRT5+ basal layer, TFAP2A+ intermediate layer, and KRT17+ periderm layer; the periderm layer is detectable only at early stages of differentiation, prior to formation of the granular and cornified layers; the periderm layer is visible in organoids around days 40–75 of differentiation. c, Overall-view image of day 70 skin cysts; the images show the major layers of skin that are required to form the skin, the epidermal and the dermal layers, and the initiating hair germs; the basal layer of skin is highlighted by KRT5+KRT15+ and CD49f+ fluorescence signals; the periderm layer is visualized by KRT15; the dermal layer (fibroblasts) is visualized by PDGFRα, and NC cell-derived mesenchymal cells within the population express P75; the SOX2+ cells represent dermal condensates at the tip of the hair germs; the initial hair placode and germ formations can be observed starting around day 55 of differentiation. PD, periderm; DC, dermal condensate. d,e, Day 75 high-magnification images representing PCAD+ hair placodes, PCAD+EDAR+LHX2+ hair germs, and PCAD+LHX2+ hair pegs; SOX2+ cells represent dermal condensates of hair germs and dermal papillae of hair pegs. f, A representative IHC image of a day 140 skin organoid. The endogenous green fluorescence from the DSP-GFP cell line visualizes epithelium of the skin cyst in the center and the hair follicles protruding from the surface of the cyst. TUJ1+ neurons are wrapping around and innervating the epithelium and the hair follicles. The skin organoids reach the lanugo-like mature stage around day 120 of differentiation. g, Representative darkfield images of a day 125 WA25 hESC-derived skin organoid (left) and dermal view of 18-week human fetal facial skin tissue (right). Skin organoids at days 120–140 resemble the mid-second trimester fetal skin tissue with (pigmented) hair follicles and adipocytes. h,i, Representative whole-mount immunostaining images of hair follicles with dermal papillae and melanocytes in a day 85 (h, left and middle) and a day 120 (i) WA25 hESC-derived skin organoids. KRT5 visualizes epithelium, outer root sheath (ORS) of hair follicles and newly forming hair germs (h, middle). SOX2 marks for melanocytes or Merkel cells present in the ORS of hair follicles and on the epithelium (h, right). MITF also specifies melanocytes in the ORS and on the epithelium. Dermal papillae of the hair follicles are also visualized by P75 (i). j, A representative whole-mount immunostaining image of a day 110 skin organoid hair follicles. The hair follicles contain a bulge region where KRT20+ touch-sensing Merkel cells are present. NEFH+ sensory neurons innervate the upper bulge region near Merkel cells. k, Representative brightfield images of plucked hairs from human fetal facial tissue at 18 weeks of gestation, adult male’s cheek (beard), skin organoid xenograft, and DSP skin organoid at day 190 of differentiation. Insets present a magnified area indicated with dash boxes. The medulla is only present in the adult beard. The medulla layer is not visible in xenograft hairs, either pigmented or non-pigmented. Darker hairs from a xenograft and a DSP skin organoid appear to contain pigmented cells that are scattered throughout the cortex, but no sign of medulla is detectable in the center of the hair shaft. See ref. 25 for additional images. The images are taken at the magnifications as follows: 200X (20X microscope objective × 10X eyepiece; b, d, e, h’); 100X (10X microscope objective × 10X eyepiece; a, c, h (left), i, and j); 50X (5X microscope objective × 10X eyepiece; g); 40X (4X microscope objective × 10X eyepiece; f and k (xenografts and organoid hairs)); 20X (2X microscope objective × 10X eyepiece; k (fetal hair and adult beard)). Scale bars, 500 μm (f, g, and k); 200 μm (a); 100 μm (b-e, and j); 50 μm (h (left) and i); 30 μm (h’).

Article Snippet: Table 2 Antibody Vendor Cat. no. RRID Host Isotope Dilution Marker for: CD49f Thermo Fisher Scientific 12-0495-81 AB_891478 https://scicrunch.org/resolver/RRID:AB_891478 Rat IgG 2a 1:50 Epidermal layer Cytokeratin 5 (KRT5) Thermo Fisher Scientific MA5-12596 AB_11008032 https://scicrunch.org/resolver/RRID:AB_11008032 Mouse IgG 1 1:50 Basal layer of skin Cytokeratin 5 (KRT5) Thermo Fisher Scientific MA5-14473 AB_10979451 https://scicrunch.org/resolver/RRID:AB_10979451 Rabbit IgG 1:50 Basal layer of skin Cytokeratin 15 (KRT15) Santa Cruz Biotechnology sc-47697 AB_627847 https://scicrunch.org/resolver/RRID:AB_627847 Mouse IgG 2a 1:50 Periderm and basal layers of skin Cytokeratin 17 (KRT17) Santa Cruz Biotechnology sc-393091 AB_2893343 https://scicrunch.org/resolver/RRID:AB_2893343 Mouse IgG 1 1:50 Periderm and basal layers of skin Cytokeratin 20 (KRT20) Cell Signaling Technology 13063 AB_2798106 https://scicrunch.org/resolver/RRID:AB_2798106 Rabbit IgG 1:100 Merkel cells E-cadherin (ECAD) BD Bioscience 610181 AB_397580 https://scicrunch.org/resolver/RRID:AB_397580 Mouse IgG 2a 1:50 Epithelia EDAR R&D Systems AF745 AB_355565 https://scicrunch.org/resolver/RRID:AB_355565 Goat IgG 1:50 Hair germs LHX2 Millipore ABE1402 AB_2722523 https://scicrunch.org/resolver/RRID:AB_2722523 Rabbit IgG 1:750 Hair germs and pegs MITF Abcam ab122982 AB_10902226 https://scicrunch.org/resolver/RRID:AB_10902226 Rabbit IgG 1:20 Melanocytes Neurofilament-heavy chain (NEFH) Cell Signaling Technology 2836 AB_10694081 https://scicrunch.org/resolver/RRID:AB_10694081 Mouse IgG 1 1:100 Sensory neurons P75NTR (P75) Cell Signaling Technology 8238 AB_10839265 https://scicrunch.org/resolver/RRID:AB_10839265 Rabbit IgG 1:50 Neural crest cells & dermal condensates P-cadherin (PCAD) Thermo Fisher Scientific 32-4000 AB_2533077 https://scicrunch.org/resolver/RRID:AB_2533077 Mouse IgG 1 1:50 Hair placodes, germs, and pegs PDGFRα (D13C6) Cell Signaling Technology 5241 AB_10692773 https://scicrunch.org/resolver/RRID:AB_10692773 Rabbit IgG 1:50 Dermal fibroblasts SCD1 Sigma-Aldrich HPA012107 AB_1856610 https://scicrunch.org/resolver/RRID:AB_1856610 Rabbit IgG 1:50 Sebaceous glands & adipocytes SOX2 BD Biosciences 561469 AB_10694256 https://scicrunch.org/resolver/RRID:AB_10694256 Mouse IgG 1 1:100 Dermal condensates and papillae, Merkel cells, Melanocytes TFAP2A DSHB 3B5 AB_528084 https://scicrunch.org/resolver/RRID:AB_528084 Mouse IgG 2b 1:5 Epithelial & neural crest cells Tubulin β class III (TUJ1) BioLegend 801202 AB_10063408 https://scicrunch.org/resolver/RRID:AB_10063408 Mouse IgG 2a 1:100 Neurons Open in a separate window Primary antibodies.

Techniques: Derivative Assay, Fluorescence, Immunostaining, Microscopy

Journal: Nature protocols

Article Title: Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

doi: 10.1038/s41596-022-00681-y

Figure Lengend Snippet: a, Schematic of mounting an immunostained organoid in a silicon isolator cassette after qCe3D clearing. A day 165 skin organoid is mounted with the qCe3D clearing solution in a well of a silicon isolator cassette. The silicone isolator is sealed with coverslips on both the top and the bottom, allowing the organoid to be imaged from both sides. b, Example of organoid imaging showing one side (left) and the other side (right) of the same organoid. KRT5 visualizes the epithelial layer of skin and ORS of hair follicles. SCD1 staining represents lipid-rich adipocytes and sebaceous glands. c, The application of the silicon isolator cassette allows for imaging up to 400 μm in depth of an organoid without loss of fluorescence signals. The panels on the right show extended depth of focus (EDF) images of individual channels of the image on the left. TUJ1 stains for neurons and KRT20 labels Merkel cells. Nuclei are visualized by DAPI. The images are taken at the magnification of 40X (4X microscope objective × 10X eyepiece; b) and 100X (10X microscope objective × 10X eyepiece; c). Scale bars, 500 μm (b); 100 μm (c).

Article Snippet: Table 2 Antibody Vendor Cat. no. RRID Host Isotope Dilution Marker for: CD49f Thermo Fisher Scientific 12-0495-81 AB_891478 https://scicrunch.org/resolver/RRID:AB_891478 Rat IgG 2a 1:50 Epidermal layer Cytokeratin 5 (KRT5) Thermo Fisher Scientific MA5-12596 AB_11008032 https://scicrunch.org/resolver/RRID:AB_11008032 Mouse IgG 1 1:50 Basal layer of skin Cytokeratin 5 (KRT5) Thermo Fisher Scientific MA5-14473 AB_10979451 https://scicrunch.org/resolver/RRID:AB_10979451 Rabbit IgG 1:50 Basal layer of skin Cytokeratin 15 (KRT15) Santa Cruz Biotechnology sc-47697 AB_627847 https://scicrunch.org/resolver/RRID:AB_627847 Mouse IgG 2a 1:50 Periderm and basal layers of skin Cytokeratin 17 (KRT17) Santa Cruz Biotechnology sc-393091 AB_2893343 https://scicrunch.org/resolver/RRID:AB_2893343 Mouse IgG 1 1:50 Periderm and basal layers of skin Cytokeratin 20 (KRT20) Cell Signaling Technology 13063 AB_2798106 https://scicrunch.org/resolver/RRID:AB_2798106 Rabbit IgG 1:100 Merkel cells E-cadherin (ECAD) BD Bioscience 610181 AB_397580 https://scicrunch.org/resolver/RRID:AB_397580 Mouse IgG 2a 1:50 Epithelia EDAR R&D Systems AF745 AB_355565 https://scicrunch.org/resolver/RRID:AB_355565 Goat IgG 1:50 Hair germs LHX2 Millipore ABE1402 AB_2722523 https://scicrunch.org/resolver/RRID:AB_2722523 Rabbit IgG 1:750 Hair germs and pegs MITF Abcam ab122982 AB_10902226 https://scicrunch.org/resolver/RRID:AB_10902226 Rabbit IgG 1:20 Melanocytes Neurofilament-heavy chain (NEFH) Cell Signaling Technology 2836 AB_10694081 https://scicrunch.org/resolver/RRID:AB_10694081 Mouse IgG 1 1:100 Sensory neurons P75NTR (P75) Cell Signaling Technology 8238 AB_10839265 https://scicrunch.org/resolver/RRID:AB_10839265 Rabbit IgG 1:50 Neural crest cells & dermal condensates P-cadherin (PCAD) Thermo Fisher Scientific 32-4000 AB_2533077 https://scicrunch.org/resolver/RRID:AB_2533077 Mouse IgG 1 1:50 Hair placodes, germs, and pegs PDGFRα (D13C6) Cell Signaling Technology 5241 AB_10692773 https://scicrunch.org/resolver/RRID:AB_10692773 Rabbit IgG 1:50 Dermal fibroblasts SCD1 Sigma-Aldrich HPA012107 AB_1856610 https://scicrunch.org/resolver/RRID:AB_1856610 Rabbit IgG 1:50 Sebaceous glands & adipocytes SOX2 BD Biosciences 561469 AB_10694256 https://scicrunch.org/resolver/RRID:AB_10694256 Mouse IgG 1 1:100 Dermal condensates and papillae, Merkel cells, Melanocytes TFAP2A DSHB 3B5 AB_528084 https://scicrunch.org/resolver/RRID:AB_528084 Mouse IgG 2b 1:5 Epithelial & neural crest cells Tubulin β class III (TUJ1) BioLegend 801202 AB_10063408 https://scicrunch.org/resolver/RRID:AB_10063408 Mouse IgG 2a 1:100 Neurons Open in a separate window Primary antibodies.

Techniques: Imaging, Staining, Fluorescence, Microscopy

Journal: Nature protocols

Article Title: Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

doi: 10.1038/s41596-022-00681-y

Figure Lengend Snippet: Primary antibodies

Article Snippet: Table 2 Antibody Vendor Cat. no. RRID Host Isotope Dilution Marker for: CD49f Thermo Fisher Scientific 12-0495-81 AB_891478 https://scicrunch.org/resolver/RRID:AB_891478 Rat IgG 2a 1:50 Epidermal layer Cytokeratin 5 (KRT5) Thermo Fisher Scientific MA5-12596 AB_11008032 https://scicrunch.org/resolver/RRID:AB_11008032 Mouse IgG 1 1:50 Basal layer of skin Cytokeratin 5 (KRT5) Thermo Fisher Scientific MA5-14473 AB_10979451 https://scicrunch.org/resolver/RRID:AB_10979451 Rabbit IgG 1:50 Basal layer of skin Cytokeratin 15 (KRT15) Santa Cruz Biotechnology sc-47697 AB_627847 https://scicrunch.org/resolver/RRID:AB_627847 Mouse IgG 2a 1:50 Periderm and basal layers of skin Cytokeratin 17 (KRT17) Santa Cruz Biotechnology sc-393091 AB_2893343 https://scicrunch.org/resolver/RRID:AB_2893343 Mouse IgG 1 1:50 Periderm and basal layers of skin Cytokeratin 20 (KRT20) Cell Signaling Technology 13063 AB_2798106 https://scicrunch.org/resolver/RRID:AB_2798106 Rabbit IgG 1:100 Merkel cells E-cadherin (ECAD) BD Bioscience 610181 AB_397580 https://scicrunch.org/resolver/RRID:AB_397580 Mouse IgG 2a 1:50 Epithelia EDAR R&D Systems AF745 AB_355565 https://scicrunch.org/resolver/RRID:AB_355565 Goat IgG 1:50 Hair germs LHX2 Millipore ABE1402 AB_2722523 https://scicrunch.org/resolver/RRID:AB_2722523 Rabbit IgG 1:750 Hair germs and pegs MITF Abcam ab122982 AB_10902226 https://scicrunch.org/resolver/RRID:AB_10902226 Rabbit IgG 1:20 Melanocytes Neurofilament-heavy chain (NEFH) Cell Signaling Technology 2836 AB_10694081 https://scicrunch.org/resolver/RRID:AB_10694081 Mouse IgG 1 1:100 Sensory neurons P75NTR (P75) Cell Signaling Technology 8238 AB_10839265 https://scicrunch.org/resolver/RRID:AB_10839265 Rabbit IgG 1:50 Neural crest cells & dermal condensates P-cadherin (PCAD) Thermo Fisher Scientific 32-4000 AB_2533077 https://scicrunch.org/resolver/RRID:AB_2533077 Mouse IgG 1 1:50 Hair placodes, germs, and pegs PDGFRα (D13C6) Cell Signaling Technology 5241 AB_10692773 https://scicrunch.org/resolver/RRID:AB_10692773 Rabbit IgG 1:50 Dermal fibroblasts SCD1 Sigma-Aldrich HPA012107 AB_1856610 https://scicrunch.org/resolver/RRID:AB_1856610 Rabbit IgG 1:50 Sebaceous glands & adipocytes SOX2 BD Biosciences 561469 AB_10694256 https://scicrunch.org/resolver/RRID:AB_10694256 Mouse IgG 1 1:100 Dermal condensates and papillae, Merkel cells, Melanocytes TFAP2A DSHB 3B5 AB_528084 https://scicrunch.org/resolver/RRID:AB_528084 Mouse IgG 2b 1:5 Epithelial & neural crest cells Tubulin β class III (TUJ1) BioLegend 801202 AB_10063408 https://scicrunch.org/resolver/RRID:AB_10063408 Mouse IgG 2a 1:100 Neurons Open in a separate window Primary antibodies.

Techniques: Isotope Dilution, Marker

Journal: Nature protocols

Article Title: Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

doi: 10.1038/s41596-022-00681-y

Figure Lengend Snippet: Primary antibodies

Article Snippet: Table 2 Antibody Vendor Cat. no. RRID Host Isotope Dilution Marker for: CD49f Thermo Fisher Scientific 12-0495-81 AB_891478 https://scicrunch.org/resolver/RRID:AB_891478 Rat IgG 2a 1:50 Epidermal layer Cytokeratin 5 (KRT5) Thermo Fisher Scientific MA5-12596 AB_11008032 https://scicrunch.org/resolver/RRID:AB_11008032 Mouse IgG 1 1:50 Basal layer of skin Cytokeratin 5 (KRT5) Thermo Fisher Scientific MA5-14473 AB_10979451 https://scicrunch.org/resolver/RRID:AB_10979451 Rabbit IgG 1:50 Basal layer of skin Cytokeratin 15 (KRT15) Santa Cruz Biotechnology sc-47697 AB_627847 https://scicrunch.org/resolver/RRID:AB_627847 Mouse IgG 2a 1:50 Periderm and basal layers of skin Cytokeratin 17 (KRT17) Santa Cruz Biotechnology sc-393091 AB_2893343 https://scicrunch.org/resolver/RRID:AB_2893343 Mouse IgG 1 1:50 Periderm and basal layers of skin Cytokeratin 20 (KRT20) Cell Signaling Technology 13063 AB_2798106 https://scicrunch.org/resolver/RRID:AB_2798106 Rabbit IgG 1:100 Merkel cells E-cadherin (ECAD) BD Bioscience 610181 AB_397580 https://scicrunch.org/resolver/RRID:AB_397580 Mouse IgG 2a 1:50 Epithelia EDAR R&D Systems AF745 AB_355565 https://scicrunch.org/resolver/RRID:AB_355565 Goat IgG 1:50 Hair germs LHX2 Millipore ABE1402 AB_2722523 https://scicrunch.org/resolver/RRID:AB_2722523 Rabbit IgG 1:750 Hair germs and pegs MITF Abcam ab122982 AB_10902226 https://scicrunch.org/resolver/RRID:AB_10902226 Rabbit IgG 1:20 Melanocytes Neurofilament-heavy chain (NEFH) Cell Signaling Technology 2836 AB_10694081 https://scicrunch.org/resolver/RRID:AB_10694081 Mouse IgG 1 1:100 Sensory neurons P75NTR (P75) Cell Signaling Technology 8238 AB_10839265 https://scicrunch.org/resolver/RRID:AB_10839265 Rabbit IgG 1:50 Neural crest cells & dermal condensates P-cadherin (PCAD) Thermo Fisher Scientific 32-4000 AB_2533077 https://scicrunch.org/resolver/RRID:AB_2533077 Mouse IgG 1 1:50 Hair placodes, germs, and pegs PDGFRα (D13C6) Cell Signaling Technology 5241 AB_10692773 https://scicrunch.org/resolver/RRID:AB_10692773 Rabbit IgG 1:50 Dermal fibroblasts SCD1 Sigma-Aldrich HPA012107 AB_1856610 https://scicrunch.org/resolver/RRID:AB_1856610 Rabbit IgG 1:50 Sebaceous glands & adipocytes SOX2 BD Biosciences 561469 AB_10694256 https://scicrunch.org/resolver/RRID:AB_10694256 Mouse IgG 1 1:100 Dermal condensates and papillae, Merkel cells, Melanocytes TFAP2A DSHB 3B5 AB_528084 https://scicrunch.org/resolver/RRID:AB_528084 Mouse IgG 2b 1:5 Epithelial & neural crest cells Tubulin β class III (TUJ1) BioLegend 801202 AB_10063408 https://scicrunch.org/resolver/RRID:AB_10063408 Mouse IgG 2a 1:100 Neurons Open in a separate window Primary antibodies.

Techniques: Isotope Dilution, Marker