The characterization of equine MSCs was performed by immunophenotyping using surface markers. The expression of CD29, CD44, CD73b, CD105, and CD45 antigens in equine adipose-derived MSCs was analyzed using primary antibodies (all purchased from Sigma-Aldrich, Munich, Germany, except for anti-CD44 obtained from R&D, Abingdon, UK) and fluorescent microscopy. Briefly, cells were fixed in 4% PFA (10 min) and then incubated in 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 h to permeabilize the cells and block nonspecific protein-protein interactions. The cells were then incubated overnight with antibodies (1:1,000 dilutions) at 4°C. Alexa Fluor 488 goat anti-rabbit (green) was used as the secondary antibody at a 1:1,000 dilution for 1 h. DAPI was used to stain the cell nuclei at a concentration of 1:1,000.
Anti cd44
Manufactured by R&D Systems
Sourced in United States, Germany, United Kingdom
Anti-CD44 is a monoclonal antibody that recognizes the CD44 cell surface glycoprotein. CD44 is a receptor for hyaluronic acid and is involved in cell-cell interactions, cell adhesion, and migration.
Automatically generated - may contain errors
Lab products found in correlation
Anti esrp1, by GeneTex (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti cd146, by R&D Systems (2 mentions)
Anti cd90, by R&D Systems (2 mentions)
Hrp conjugated secondary antibody, by Cytiva (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti snail1, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Gapdh, by GE Healthcare (1 mentions)
Anti γ catenin, by Cell Signaling Technology (1 mentions)
C2 laser scanning confocal microscope, by Nikon (1 mentions)
Canto 2, by BD (1 mentions)
Anti stro 1, by R&D Systems (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Hyaluronan, by Lifecore Biomedical (1 mentions)
Anti zeb1, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Ecl prime blocking agent, by GE Healthcare (1 mentions)
Hsp90 antibody, by Cell Signaling Technology (1 mentions)
15 protocols using anti cd44
1
Characterization of Equine Adipose-Derived MSCs
2
Western Blot Protein Detection Protocol
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We performed Western blotting, involving detection of protein bands as enhanced chemo-luminescence signal on X-ray films, as described previously [25 (link)]. The following primary antibodies were used for protein detection: anti-METTL3 (catalog number PA5-28178 ThermoFisher Scientific, Waltham, MA, USA), anti-ESRP1 (catalog number GTX131373, GeneTex, Irvine, CA, USA), (catalog number ab155227, Abcam, Cambridge, MA, USA), anti-CD44 (catalog number MAB7045, R&D Systems, Minneapolis, MN, USA), anti-SNAIL1 (catalog number 3895, Cell Signaling Technology, Danvers, MA, USA), and anti-vimentin (catalog number 3932, Cell Signaling Technology). Each Western blot was performed at least twice; representative blots are shown. We quantified relative intensities of protein bands detected on X-ray films with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Analyzing Epithelial-Mesenchymal Transition Markers
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Cells were lysed in RIPA buffer and analyzed by Western blotting as described (Huang et al. 2014 ). Antibodies used for Western blotting were as follows: anti-CD44 (R&D Systems), anti-hnRNPF (Santa Cruz Biotechnology), anti-E-cadherin (Cell Signaling Technology), anti-γ-catenin (Cell Signaling Technology), and anti-vimentin (NeoMarkers). GAPDH (GE) was used as a loading control. For immunofluorescence, cells were fixed in 4% polyformaldehyde and permeabilized with 0.2% Triton X-100 before blocking with 5% BSA. Primary antibody incubations with rabbit anti-E-cadherin (1:200; Cell Signaling Technology) were performed overnight at 4°C followed by incubation with Alexa fluor 488-conjugated anti-rabbit IgG (1:500; Invitrogen) for 2 h at room temperature and DAPI staining. Representative images were captured under a Nikon C2 laser scanning confocal microscope.
4
Stem Cell Immunophenotyping by Flow Cytometry
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The percentage of surface markers indicating the stem cell immunophenotype was analyzed by flow cytometry. The PDLSCs, PULPSCs, BMSCs, and CDCs from each group were harvested in PBS, washed once with PBS, fixed in 3.7% paraformaldehyde (Wako, Tokyo, Japan), and then incubated with anti-Stro-1, anti-CD44, anti-CD90, and anti-CD146, antibodies (R&D Systems, Minneapolis, MN, USA) at a dilution of 1:100 (volume/volume) in PBS for 1 hour at room temperature in the dark. Immunostaining with an antibody against the hematopoietic marker anti-CD19 and anti-CD45 was conducted as a negative control. The cells were then incubated with secondary anti-mouse antibodies—allophycocyanin-conjugated antibody for CD44, CD90, CD146, CD19 and CD45 or phycoerythrin-conjugated antibody for STRO-1—at a dilution of 1:100 (volume/volume) in PBS for 30 minutes at 4°C in the dark, and the staining was finally analyzed with a flow-cytometry cell-sorting device (CANTO II, Becton Dickinson, Franklin Lakes, NJ, USA) [30 (link)].
5
CD44-Mediated Hyaluronan Signaling
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Anti-CD44 was obtained from R&D systems, UK. HRP-conjugated secondary antibodies were obtained from Amersham, UK and Hyaluronan (MW220 kDa) from Lifecore Biomedical (MN, USA). Cells were stimulated with 100 μg/ml HA for indicated times. All other reagents were purchased from Sigma (Poole, UK) unless otherwise stated.
6
CD44 Protein Immunodetection in Hippocampal CA3
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The procedure was performed in the hippocampal CA3 field from postnatal day 53 (P53) rat brain as described previously (Wilczynski et al., 2008 (link)). For immunodetection, sheep polyclonal anti-CD44 (1:50; R&D Systems, Minneapolis, MN) followed by secondary antibodies coupled to 10-nm gold particles was applied (Electron Microscopy Sciences, Fort Washington, PA). Nonimmune sheep IgG (BD PharMingen, San Jose, CA) was used as a negative control. Gold particle densities within various ultrastructural compartments were measured on digital micrographs using ImageJ software (National Institutes of Health, Bethesda, MD). The statistical evaluation of the labeling was performed using χ2 test of observed and expected gold counts over the given compartments (Mayhew and Lucocq, 2008 (link)). The data were sampled randomly. At least 200 gold particles at ∼10 images from three animals per group were counted.
7
Comprehensive Western Blotting Approach
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We performed western blotting as described previously [39 (link)]. The following primary antibodies were used for protein detection: anti-TET2 (catalog number 61390, Active Motif, Carlsbad, CA), anti-ESRP1 (catalog number GTX131373, GeneTex, Irvine, CA, USA), anti-CD44 (catalog number MAB7045, R&D Systems, Minneapolis, MN, USA), anti-ADARB2 (catalog number sc-73410, Santa Cruz Biotechnology, Dallas, TX, USA), anti-ZEB1 (catalog number 3396, Cell Signaling Technology, Danvers, MA, USA), anti-GRHL2 (catalog number HPA004820, Sigma-Aldrich, St. Louis, MO), and anti-vimentin (catalog number 3932, Cell Signaling Technology). The ECL prime blocking agent (GE Healthcare Life Sciences, Piscataway, NJ, USA) was used for blocking and Lumigen TMA-6 reagents for detection (Lumigen, Inc., Southfield, MI, USA). The blots were re-probed with an HSP90 antibody (catalog number 4875, Cell Signaling Technology) or with an anti-β-actin antibody (catalog number A5316, Sigma-Aldrich) to document equal protein loading. Each western blot was performed at least twice; representative blots are shown. Relative intensities of the bands were determined with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
8
Intravitreal Injection Technique for Ocular Research
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The pupil of the anesthetized eye was dilated with tropicamide drops. A 32-gauge needle was inserted 2 mm behind the temporal limbus and directed toward the optic nerve to make an access for drug delivery. Subsequently, one dose of 2 μl OPN (500ng/ml, diluted in PBS; Merck KGaA, Darmstadt, Germany), OPN neutralizing antibody (Anti-OPN, 50 μM, diluted in PBS; Merck KGaA), CD44 neutralizing antibody (Anti-CD44, 20μg/ml, diluted in PBS, R&D systems China Co. Ltd), Selective inhibitor of ItgαVβ3 (cyclo RGDyk, 20nM, diluted in 0.2% DMSO, Selleckchem, Houston, TX, USA), minocycline (50 μM, diluted in PBS, Sigma-Aldrich, St. Louis, MO, USA) or SB203580 (50 μM, diluted in 0.2% DMSO; Sigma Aldrich) was injected into the vitreous cavity through the access using a microinjector (Hamilton Bonaduz AG, Switzerland) under a stereoscopic microscope (Carl Zeiss Microscopy, Jena, Germany) according to experimental needs. Each eye that received only an injection of PBS or 0.2% DMSO in the same manner served as a negative control.
9
Phenotypic Characterization of MSCs
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MSCs were incubated with anti-CD19, anti-CD146, anti-CD44, anti-CD45, anti-CD90, and anti-CD105 antibodies (R&D, USA) at room temperature for 30 min. After washing twice with PBS, the MSCs were incubated with a FITC-labeled secondary antibody in the dark for 30 min. After washing, the cells were suspended in PBS and analyzed on a flow cytometer.
10
Protein Expression Analysis in Cells
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Whole cell lysates were obtained with radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA). The protein concentrations were measured using a BCA Protein Assay Kit (Pierce, Rockfold, IL). The following antibodies were used: anti-FGFR4, anti-pFRS2α, anti-pSTAT3, anti-pAKT, anti-pERK, and anti-Snail from Cell Signaling Technology (Danvers, MA); anti–E-cadherin from BD Sciences (San Jose, CA); anti-vimentin from Santa Cruz Biotechnology (Santa Cruz, CA); anti–β-actin and anti-Twist from Abcam (Cambridge, UK); anti-CD133 from Miltenyi Biotec (Bergisch Gladbach, Germany); and anti-CD44 from R&D Systems (Minneapolis, MN).
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