Human fetal gonads were dissociated in 0.25% trypsin-EDTA (Gibco) at 37° C for 20 min and collected by centrifugation at 2000 rpm in a microcentrifuge. Dissasociated cells were resuspended in a buffer containing 1% FBS (Gibco). cKit+/CD117+ cells were selected by using an immunomagnetic separation system (MiniMACS, 130-091-332, Miltenyi Biotec). Purification efficiency (higher than 90%) was verified by alkaline phosphatase staining (SK-5100, Vector Laboratories) (Suppl Fig 1 ).
Sk 5100
Manufactured by Vector Laboratories
Sourced in United States
The SK-5100 is a high-performance liquid chromatography (HPLC) system designed for efficient and accurate sample analysis. It features a modular design, allowing for customization to meet specific laboratory requirements. The core function of the SK-5100 is to separate, identify, and quantify components in a liquid sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse cd326 epcam, by BioLegend (2 mentions)
Anti mouse ssea1 apc 647, by BioLegend (2 mentions)
Anti mouse cd90 thy1.2 efluor 450, by Thermo Fisher Scientific (2 mentions)
Ak 5000, by Vector Laboratories (2 mentions)
Ba 4001, by Vector Laboratories (2 mentions)
Gtx 76011, by GeneTex (2 mentions)
Mca1211, by Bio-Rad (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Minimacs, by Miltenyi Biotec (1 mentions)
Sc 15334, by Santa Cruz Biotechnology (1 mentions)
Click it edu alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Normal blocking serum, by Vector Laboratories (1 mentions)
Mab377, by Merck Group (1 mentions)
Ab3623, by Merck Group (1 mentions)
Axioskop 40, by Zeiss (1 mentions)
Kaiser s glycerol gelatin, by Merck Group (1 mentions)
Kaiser s glycerin gelatine, by Merck Group (1 mentions)
Fluorophore conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
9 protocols using sk 5100
1
Enrichment of Human Fetal cKit+ Cells
2
Quantifying Proliferation and Mucin Expression
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Living cells were pulsed with 5-ethynyl-2-deoxyuridine (EdU) and assayed for alkaline phosphatase (ALP). The cells then were fixed and sequentially stained for EdU incorporation (S-phase cells), mucin 2, and DNA. Cells were first pulsed with 10 μmol/L of EdU for 24 hours at 37°C, rinsed with PBS, and incubated with a red ALP substrate (SK-5100; Vector Laboratories, Burlingame, CA) in Tris buffer (0.15 mol/L, pH 8.4) for 30 minutes at 37°C. The cells were rinsed with PBS, fixed in 4% paraformaldehyde for 15 minutes, and permeabilized with 0.5% Triton X-100 in PBS for 20 minutes. Incorporated EdU was labeled with Click-iT EdU Alexa Fluor 647 (C10340; ThermoFisher). The cells were then rinsed with 0.75% glycine in PBS for 5 minutes 3 times, followed by blocking with 10% donkey serum (017-000-121; Jackson Immunoresearch, West Grove, PA) for 1 hour. The cells were incubated in rabbit mucin 2 (Muc2) antibody (1:200, sc-15334; Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight, and stained with donkey anti-rabbit IgG-conjugated Alexa Fluor 488 (1:500, 711-545-152; Jackson Immunoresearch) for 45 minutes. Finally, the DNA was stained with Hoechst 33342 (2 μg/mL, B2261; Sigma-Aldrich) for 15 minutes.
3
Reprogramming Refractory MEFs to iPSCs
4
Dual Immunohistochemical Labeling Protocol
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Brain sections were immersed in 3% H2O2/methanol for 15 min and then incubated with a diluted normal blocking serum (Vector Laboratories, CA, USA) at RT for 25 min. Sections were then incubated with a mouse antineuronal nuclei (NeuN) antibody (1:200 dilution, MAB 377 Chemicon) 1.5 h at 37°C and washed with DPBS. Following their incubation with the diluted biotinylated secondary antibody and an ABC-AP reagent (AK-5002, Vectastain), the sections were stained with an alkaline phosphatase substrate solution (SK-5300, Vector Blue). They were then incubated with a rabbit anti-active caspase-3 antibody (17 kD, 1:100 dilution, AB3623 Chemicon) for 1.5 h at 37°C and washed with DPBS. Following their incubation with the diluted biotinylated secondary antibody and an ABC-AP reagent (AK-5001, Vectastain), the sections were stained with an alkaline phosphatase substrate solution (SK-5100, Vector Red), dried, and mounted in mounting media (Assistant-Histokitt, Germany). Finally, the immunopositive cells were detected using microscopic analysis (Axioskop 40, Zeiss).
5
Immunohistochemical Quantification of Platelets
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Myocardial tissue was embedded with O.C.T., cut into 10-μm-thick sections, and fixed with acetone. For staining of platelets, a rat anti-mouse CD41 antibody was used (GTX 76011, GeneTex, Irvine, CA), and for control purposes, an IgG1 isotype control was used (MCA1211, Serotec, Puchheim, Germany). Secondary staining was performed with a biotinylated rabbit anti-rat IgG (BA-4001, Vector). Alkaline phosphatase and substrate kit (AK-5000 & SK-5100, Vector) and levamisole (X3021, DAKO) were used for detection. Finally, samples were embedded with Kaiser’s glycerol gelatin (Merck, Darmstadt, Germany) until adequate staining.
Platelets were counted in two representative pictures of × 20 magnification out of the inflamed myocardium of two different CD41–stained slices. For counting MPIOs, the inflamed myocardium of 10 representative CD41 stained slices was examined carefully.
Platelets were counted in two representative pictures of × 20 magnification out of the inflamed myocardium of two different CD41–stained slices. For counting MPIOs, the inflamed myocardium of 10 representative CD41 stained slices was examined carefully.
6
Reprogramming Refractory MEFs to iPSCs
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Reprogrammable MEFs grown in mESC media were dox-treated for 5 days, then harvested and stained for various surface markers: Anti-mouse CD326 EPCAM (PE), anti-mouse SSEA1 APC-647 (Biolegend) and anti-mouse CD90 THY1.2 eFluor 450 (eBiosciences). THY1+/SSEA1-/EPCAM-/OCT4-GFP-cells (“refractory cells”) were sorted in equal numbers onto gelatin-coated 6-well plates containing mESC media containing doxycycline, doxycycline+ascorbic acid, doxycycline+GSK3i or doxycycline+AGi. Media and small molecules were replaced every other day, for a total of 7 days, after which dox and AGi were withdrawn and replaced with fresh mESC media. Dox-independent iPSCs were scored at least three days later by alkaline phosphatase staining kit (Vector Labs SK-5100).
7
Quantifying Pulmonary Embolism and Microparticles
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Platelets were stained using a rat anti-mouse CD41 antibody (GTX 76011, GeneTex, Irvine/CA, USA) or unspecific control IgG1 isotype (MCA1211, Serotec, Puchheim, Germany). For secondary staining a biotinylated rabbit anti-rat IgG (BA-4001, Vector, Burlingame/CA, USA) was used. Afterwards, alkaline phosphatase and substrate kit (AK-5000 & SK-5100; Vector, Burlingame/CA, USA) after levamisol pre-treatment (X3021, DAKO, Hamburg, Germany) was applied for antibody detection. Sections were then embedded in Kaiser’s Glyceringelatine (1092420100, Merck, Hamburg, Germany). Due to their size (1 μm), MPIO beads are visible using 63x magnification without further staining. MPIOs were quantified as mean number of MPIOs of 3 sections per section at 63x magnification.
Histology sections were analyzed using a light microscope (Zeiss optics, Germany). Pulmonary embolism was quantified as mean of 3 representative sections of the according. Pulmonary emboli were graded according to size of obstructed vessels: small vessel (maximum diameter < 150 μm: GI < 50% vessel obstruction, GII > 50% vessel obstruction) or large vessels (maximum diameter > 150 μm: GIII < 50% vessel obstruction, GIV > 50% vessel obstruction).
Histology sections were analyzed using a light microscope (Zeiss optics, Germany). Pulmonary embolism was quantified as mean of 3 representative sections of the according. Pulmonary emboli were graded according to size of obstructed vessels: small vessel (maximum diameter < 150 μm: GI < 50% vessel obstruction, GII > 50% vessel obstruction) or large vessels (maximum diameter > 150 μm: GIII < 50% vessel obstruction, GIV > 50% vessel obstruction).
8
Immunohistochemical Analysis of DCLK1+ Cells
9
Alkaline Phosphatase Staining of Fisetin-Treated Cells
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Cells were cultured in a 35 mm tissue culture dish with and without fisetin supplementation for 21 days. The cells were then stained with an alkaline phosphatase staining kit according to the manufacturer’s protocol (SK-5100; Vector Laboratories, Burlingame, CA, USA).
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