Promoting Osseointegration of Ti Implants through Micro/Nano-Scaled Hierarchical Ti Phosphate / Ti Oxide Hybrid Coating
|
|
- Neal Day
- 5 years ago
- Views:
Transcription
1 Promoting Osseointegration of Ti Implants through Micro/Nano-Scaled Hierarchical Ti Phosphate / Ti Oxide Hybrid Coating Nan Jiang 1, Zhijun Guo 2,3, Dan Sun 3, Yubao Li 2, Yutao Yang 1, Chen Chen 2, Li Zhang *2 and Songsong Zhu *1 1. State Key Laboratory of Oral Diseases, & National Clinical Research Center for Oral Disease, & West China Hospital of Stomatology, Sichuan University, Chengdu, China 2. Research Center for Nano-Biomaterials, Analytical & Testing Center, Sichuan University, Chengdu, China 3. School of Mechanical & Aerospace Engineering, Queens University Belfast, Belfast, UK Materials and methods Cell isolation and culture Bone marrow stromal cells (BMSCs) were isolated from the femurs of 4-week-old male Sprague-Dawley rats (Animal Research Center, Sichuan University, China) and subsequently cultured in alpha-modified Eagle s medium (α-mem, Gibco, Gaithersburg, MD, USA), which was supplemented with 10% fetal bovine serum (FBS, Gibco), 50 mg/l ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 10 mm Na-β-glycerophosphate (Sigma-Aldrich), 10 8 M dexamethasone (Sigma-Aldrich), and 1% penicillin-streptomycin antibiotic antimycotic solution (Invitrogen, Carlsbad, CA, USA). The cells were incubated in a humidified atmosphere (95% air and 5% CO 2 ) at 37 C. After 48 h, the unattached cells were removed by rinsing, and fresh culture medium was topped up. The culture medium was replaced every 2 3 days, and the cells were sub-cultured when reaching 80 90% confluence.
2 Cell cycle analysis A 5'-bromo-2'-deoxyuridine (BrdU) flow kit (BD Pharmingen, San Diego, CA) was used to determine the cell cycle kinetics and to measure the incorporation of BrdU into DNA of the proliferating cells. The assay was performed according to the manufacturer's protocol. Briefly, cells (1x10 6 /well) were seeded overnight in 24-well tissue culture plates for 3 days, followed by the addition of 10 µm BrdU, and the incubation was continued for an additional 4 h. Cells were pooled from triplicate wells for every sample group, fixed in a solution containing paraformaldehyde and the detergent saponin, and incubated for 1 h with DNase at 37 C (30 µg per sample). FITC-conjugated anti-brdu antibody (1:50 dilution in wash buffer; BD Pharmingen) was added and incubation was continued for 20 min at room temperature. Cells were then rinsed and DNA was stained using 7-aminoactinomycin D (7-AAD; 20 µl per sample). Data collection and analyses were performed using an Accuri C6 flow cytometer (BD Biosciences). Samples were gated on SSC-A vs. FSC-A. Approximately 10,000 gated events were collected. The BrdU content (FITC) and total DNA content (7-AAD) were determined using FlowJo 10 software (De Novo Software). ALP activity assay Cell differentiation potential on the Ti disc samples was evaluated by alkaline phosphatase (ALP) activity. After incubation with the samples for 24 h, the culture medium was changed to osteogenic inductive medium, containing α-mem, 10% FBS, 100 U/mL penicillin, 100 mg/ml streptomycin sulfate, 100 nm dexamethasone, 50 µg/ml ascorbic acid, and 10 mm Na-β-glycerophosphate. The osteogenic inductive medium was replaced every 2 days. After
3 incubation for 7, 10, and 14 days, the cells seeded on all the Ti samples were fixed by 4% paraformaldehyde (Sigma-Aldrich) and stained with an ALP kit (Beyotime, Shanghai, China). Then the cells were incubated with p-nitrophenyl phosphate (pnpp, Sigma-Aldrich) for 30 min, and ALP activity was evaluated through measurement of optical density using the spectrophotometer at 405 nm. The total protein content was calculated employing Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) and normalized relative to bovine serum albumin (BSA, Sigma-Aldrich) at 630 nm. A total of 4 samples per coating composition were examined at each time point, and the final results are shown as mean values ± standard deviations. Western blot assay Seeded at a density of cells/well, the BMSCs were cultured on Ti samples for 48 h. The cells were then trypsinized and lysed in the RIPA buffer (Roche, Switzerland). The protein concentration in the extracted lysates was determined using a BCA protein assay kit (Beyotime, China). The samples were separated by SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad, USA). The membrane was then blocked with 5% BSA (Gibco, USA) in the TTBS solution. The samples were then incubated with anti-integrinβ1, anti-vinculin, anti-runx-2, and anti-opn primary antibody (1:500 dilution; Abcam, UK) and followed by horseradish peroxidase (HRP) conjugated secondary antibody (1:3500 dilution; CST, USA) for 60 min at room temperature. The intensity of the protein bands were quantified on a Western-Light Chemiluminescent Detection System.
4 Quantitative real-time PCR analysis After incubation with the Ti disc samples for 24 h and 48 h, the cells were collected for qrt-pcr test to explore RNA expression of the following factors: integrinβ1, vinculin, runt-related transcription factor 2 (Runx-2), collagen type I (Col-I), osteopontin (OPN) and osteocalcin (OCN). After the cells were detached by 0.25% trypsin-1 mm EDTA (Gibco BRL, Gaithersburg, MD, USA), total RNA was extracted employing TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then the mrna was reversely transcribed to cdna with the aid of a first-strand cdna synthesis kit (Takara, Shiga, Japan). After that, the relative expression levels of the osteogenic factors were determined using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). GAPDH was used as the internal RNA control and the primer sequences were listed in Supplementary Table S1. Animal model The in vivo animal study was conducted in accordance with international standards on animal welfare as well as the Animal Research Committee of the university. A total of 40 Sprague-Dawley rats (age 3 months, weight g, Animal Research Center, Sichuan University, China) were used in the present study. The rats were placed under general anesthesia with abdominal injection of ketamine (100 mg/kg, Yuhan, Seoul, Korea) and xylazine (10 mg/kg, Bayer, Ansan, Korea). Bilateral longitudinal incisions (length: 10 mm) were made along the medial side of the knee joint, and a 1-mm hole was drilled using an inter-condylar notch, cooling with sterile saline solution. The rats were randomly assigned into four groups (Group A, B, C, and D, n=10 animals per group) and inserted with the
5 titanium implants (n=20 samples per group) as follows: Group A (cp-ti), Group B (P-C-Ti), Group C (P-G-Ti), and Group D (P-R-Ti). The implantation was made into the medullary canal of the metaphysis until the implant reached the articular surface, and the patella was relocated, reconstructing the extensor mechanism. The rats received intramuscular antibiotic and analgesic injection for 3 days and were allowed for free activities ad libitum. Twelve weeks following the surgery, the rats were sacrificed and the proximal tibiae (together with the titanium implant) were harvested for the following evaluations. Micro-CT evaluation 12 weeks after surgery, the specimens (n=10 per group) were harvested and surveyed by a high resolution µ-ct scanner system (Scanco Medical µ-ct 50, Switzerland). For the best X-ray scanning effect, the parameter of voltage, electric current, and integration time was set at 70 kv, 120µA and 700ms, respectively. The multi-level thresholds were applied and the reconstructive layer thickness was set to 10µm. To distinguish the implant from the bone, the threshold value was set to In peripheral quantitative evaluation, the constrained Gaussian filter value was maintained at σ = 1. 2 and support = 1. Histological analysis 12 weeks after the surgery, undecalcified dissections were conducted for the other half tibiae with implants (n=10 per group) The samples were fixed with 4% formalin buffered solution, rinsed by water and progressively dehydrated in ethanol with different concentrations (20%, 40%, 60%, 75%, 90%, and 100%). After being immobilized in poly-methyl-methacrylate
6 resin, the specimens were sectioned longitudinally into 80 µm thick slices using a rotary cutting saw (SP1600, Leica, Germany) and polished down to ~ 50µm using a polishing machine (Buelher Metaserv 2000). 1% toluidine blue was used to stain the trabecula bone for observation in VOI. The histological evaluation was carried out on sections approximately 1mm below the growth plate using a Leica DM-RBE microscope (Leica, Germany). Bone area ratio (BA), defined as the percentage of mature bone to the whole tissue region within a ring area extending 250 µm from the implant surface; and bone-to-implant contact (BC), defined as the percentage of the linear fraction of mineralized bone in direct contact with the implant interface, were calculated with the aid of the NIS-Elements F2.20 image software (Media Cybernetics, USA). Table S1. Primers Designed for Each Targeted Genes. Gene Forward primer Reverse primer Integrinβ1, GTGCCTCTTGACTACACGACC GGCGGAAAGGGTTATCCTTCA Vinculin TCTCGCACCTGGTGATTATGC TGAACAGTCTCTTTTCCAACCC Runx-2 CAGGCGTATTTCAGATGATGACA TAAGTGAAGGTGGCTGGATAGTG Col-I AACAGTCGCTTCACCTACAG AATGTCCAAGGGAGCCAC OPN CTACGACCATGAGATTGGCAG CATGTGGCTATAGGATCTGGG OCN GGAGGGCAGTAAGGTGGTGA ACGGTGGTGCCATAGATGC GAPDH AGGTCGGTGTGAACGGATTTG TGTAGACCATGTAGTTGAGGTCA
7 Figure S1. Quantification of protein adsorption area on each sample Figure S2. A: Flow cytometry analysis for cell proliferation. SSC and FSC showed the target cells are almost identical, B: Target cells in each group, C: Quantitative analysis of S phase cells proportion in different samples. *p < 0.05 vs cp-ti.
8 Figure S3. The integrinβ1 and vinculin expressions of BMSCs after 48h culture in each group observed by CLSM. The magnification of the images is 800. Figure S4. A: ALP stain for BMSCs on day 7 for each group, B: The relative ALP activity of BMSCs incubated on different samples for 1,3 and 7days. *p < 0.05 vs cp-ti, #p < 0.05 vs P-C-Ti, ^p < 0.05 vs P-G-Ti.
9 Figure S5. The OCN and OPN expression of BMCSs after 48h culture in each group investigated by CLSM. The magnification of the images is 800.
10 Figure S6. A: Insertion of implants; B: the position of the implant in the tibia.
A Supersandwich Fluorescence in Situ Hybridization (SFISH) Strategy. for Highly Sensitive and Selective mrna Imaging in Tumor Cells
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2015 Electronic Supplementary Information (ESI) A Supersandwich Fluorescence in Situ Hybridization (SFISH)
More informationFor Western blot analyses, cells were lysed in RIPA buffer (50 mm Tris-HCl ph 7.2,
Western blot assay For Western blot analyses, cells were lysed in RIPA buffer (50 mm Tris-HCl ph 7.2, 150 mm NaCl, 1% NP40, 0.1% SDS, 0.5% DOC, 1 mm PMSF, 25 mm MgCl 2, and supplemented with a phosphatase
More informationSupplemental methods:
Supplemental methods: ASC-J9 treatment ASC-J9 was patented by the University of Rochester, the University of North Carolina, and AndroScience Corp., and then licensed to AndroScience Corp. Both the University
More informationDNA methylation analysis was carried out using the Epityper system from Sequenom
Supplemental methods Quantitative DNA methylation analysis DNA methylation analysis was carried out using the Epityper system from Sequenom (San Diego, CA). The EpiTYPER assay is a tool for the detection
More informationEffects of Tetrahedral DNA Nanostructures on Autophagy in. Chondrocytes
Electronic Supplementary Material (ESI) for Chemical Communications. This journal is The Royal Society of Chemistry 2018 Effects of Tetrahedral DNA Nanostructures on Autophagy in Chondrocytes Sirong Shi,
More informationMarilyn G. Rimando, Hao-Hsiang Wu, Yu-An Liu, Chien-Wei Lee, Shu-Wen Kuo, Yin-
Supplementary Information Glucocorticoid receptor and Histone Deacetylase 6 mediate the differential effect of dexamethasone during osteogenesis of Mesenchymal stromal cells (MSCs) Marilyn G. Rimando,
More informationApoptosis And Anti-tumor Effect Induced By Mtor Inhibitor And Autophagy Inhibitor In Human Osteosarcoma Cells
Apoptosis And Anti-tumor Effect Induced By Mtor Inhibitor And Autophagy Inhibitor In Human Osteosarcoma Cells Ryosuke Horie. Kagawa University of medecine, Kita-gun, Japan. Disclosures: R. Horie: None.
More informationSupplemental Information. Materials and methods.
Supplemental Information Materials and methods. Cell culture. hmscs were isolated from bone marrow of 3 male donors, undergoing orthopedic surgery (mean age 69.7). Cells were cultured in high glucose DMEM
More informationSupplementary Information Temperature-responsive Gene Silencing by a Smart Polymer
Supplementary Information Temperature-responsive Gene Silencing by a Smart Polymer Mingming Wang, Yiyun Cheng * Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal
More informationRNA oligonucleotides and 2 -O-methylated oligonucleotides were synthesized by. 5 AGACACAAACACCAUUGUCACACUCCACAGC; Rand-2 OMe,
Materials and methods Oligonucleotides and DNA constructs RNA oligonucleotides and 2 -O-methylated oligonucleotides were synthesized by Dharmacon Inc. (Lafayette, CO). The sequences were: 122-2 OMe, 5
More informationSegments of the obstructed intestinal loops were fixed in 4% paraformaldehyde
Supplementary text Supplementary materials and methods Histopathological examination Segments of the obstructed intestinal loops were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin wax with
More informationSpironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice
Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice Supplementary Material Supplementary Methods Materials Spironolactone, aldosterone and β-glycerophosphate were
More informationInduction of Neurogenesis in Rat Bone Marrow Mesenchymal Stem Cells Using Purine Structure- Based Compounds
Electronic supplementary information (ESI) Induction of Neurogenesis in Rat Bone Marrow Mesenchymal Stem Cells Using Purine Structure- Based Compounds Mi Hee Cho, Jung Hwa Lee, Hyun Hee Ahn, Ju Young Lee,
More informationVDL602.2 RAPID ASSAY FOR DETERMINING ADENOVIRAL VECTOR TITER
1. Purpose 1.1. The purpose of this protocol is to determine the number of infectious adenoviral particles. 1.2. The starting material is purified adenoviral vectors. 1.3. This protocol is based upon the
More informationSupplementary Table 1. PCR amplification conditions for each primer pair. Primer sequence
- 1 - Supplementary Tables Supplementary Table 1. PCR amplification conditions for each primer pair Primer sequence FN1 S - CAAAGCAAGCCCGGTTGT AS - CGCTCCCACTGTTGATTTATCTG ITGα2 S - TTAGGTTACTCTGTGGCTGCAATT
More informationB. ADM: C. D. Apoptosis: 1.68% 2.99% 1.31% Figure.S1,Li et al. number. invaded cells. HuH7 BxPC-3 DLD-1.
A. - Figure.S1,Li et al. B. : - + - + - + E-cadherin CK19 α-sma vimentin β -actin C. D. Apoptosis: 1.68% 2.99% 1.31% - : - + - + - + Apoptosis: 48.33% 45.32% 44.59% E. invaded cells number 400 300 200
More informationApoptosis assay: Apoptotic cells were identified by Annexin V-Alexa Fluor 488 and Propidium
Apoptosis assay: Apoptotic cells were identified by Annexin V-Alexa Fluor 488 and Propidium Iodide (Invitrogen, Carlsbad, CA) staining. Briefly, 2x10 5 cells were washed once in cold PBS and resuspended
More informationSupplemental Materials and Methods
Supplemental Materials and Methods In situ hybridization In situ hybridization analysis of HFE2 and genin mrna in rat liver tissues was performed as previously described (1). Briefly, the digoxigenin-labeled
More informationIn vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells Ningfei An, Yubin Kang *
In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells Ningfei An, Yubin Kang * Division of Hematology-Oncology, Department of Medicine, Medical University of South Carolina, Charleston,
More informationIsolation, culture, and transfection of primary mammary epithelial organoids
Supplementary Experimental Procedures Isolation, culture, and transfection of primary mammary epithelial organoids Primary mammary epithelial organoids were prepared from 8-week-old CD1 mice (Charles River)
More informationLINGO-1, A TRANSMEMBRANE SIGNALING PROTEIN, INHIBITS OLIGODENDROCYTE DIFFERENTIATION AND MYELINATION THROUGH INTERCELLULAR SELF- INTERACTIONS.
Supplemental Data: LINGO-1, A TRANSMEMBRANE SIGNALING PROTEIN, INHIBITS OLIGODENDROCYTE DIFFERENTIATION AND MYELINATION THROUGH INTERCELLULAR SELF- INTERACTIONS. Scott Jepson, Bryan Vought, Christian H.
More informationReprogramming of Dermal Fibroblasts into Osteo-Chondrogenic Cells
Stem Cell Reports, Volume 8 Supplemental Information Reprogramming of Dermal Fibroblasts into Osteo-Chondrogenic Cells with Elevated Osteogenic Potency by Defined Transcription Factors Yinxiang Wang, Ming-Hoi
More informationsirna Transfection Into Primary Neurons Using Fuse-It-siRNA
sirna Transfection Into Primary Neurons Using Fuse-It-siRNA This Application Note describes a protocol for sirna transfection into sensitive, primary cortical neurons using Fuse-It-siRNA. This innovative
More informationSupplementary Information (Ha, et. al) Supplementary Figures Supplementary Fig. S1
Supplementary Information (Ha, et. al) Supplementary Figures Supplementary Fig. S1 a His-ORMDL3 ~ 17 His-ORMDL3 GST-ORMDL3 - + - + IPTG GST-ORMDL3 ~ b Integrated Density (ORMDL3/ -actin) 0.4 0.3 0.2 0.1
More informationTumor tissues or cells were homogenized and proteins were extracted using
SUPPLEMENTAL MATERIALS AND METHODS Western Blotting Tumor tissues or cells were homogenized and proteins were extracted using T-PER tissue protein extraction buffer. Protein concentrations were determined
More informationMethods Western blot analysis of plg Quantification of plasminogen accumulation by ELISA Immunohistochemical analysis
Methods Western blot analysis of plg Wild-type mice first received a standardized burn wound and then were intravenously administered 2 mg of human plg (Omnio AB, Umeå, Sweden). 24 hours after wounding
More informationSupplemental Information
Supplemental Information RSPO3-LGR4 regulates osteogenic differentiation of human adipose-derived stem cells via ERK/FGF signaling Min Zhang a,b1, Ping Zhang a,b1, Yunsong Liu a,b, Longwei Lv a,b, Xiao
More information< Supporting Information >
SUPPORTING INFORMATION 1 < Supporting Information > Discovery of autophagy modulators through the construction of high-content screening platform via monitoring of lipid droplets Sanghee Lee, Eunha Kim,
More informationThe Healing Of Tibial And Calvarial Defect Using Runx2-transfected Adipose Stem Cells
The Healing Of Tibial And Calvarial Defect Using Runx2-transfected Adipose Stem Cells Jong-Min Lee, Ph.D. 1, Eun-Ah Kim, MS 2, Gun-Il Im, MD 2. 1 Dongguk University, Goyang, Korea, Republic of, 2 Dongguk
More informationWestern Blot Tool Box
Western Blot Tool Box BOX12/BOX12-03 V1.1 Store at 2-8 C For research use only Introduction The Western Blot Tool Box is designed to conveniently provide reagents/buffers needed for Western blotting, from
More informationSupplementary Figure 1. Co-localization of GLUT1 and DNAL4 in BeWo cells cultured
Supplementary Figure 1. Co-localization of GLUT1 and DNAL4 in BeWo cells cultured under static conditions. Cells were seeded in the chamber area of the device and cultured overnight without medium perfusion.
More informationSupplementary Information: Materials and Methods. Immunoblot and immunoprecipitation. Cells were washed in phosphate buffered
Supplementary Information: Materials and Methods Immunoblot and immunoprecipitation. Cells were washed in phosphate buffered saline (PBS) and lysed in TNN lysis buffer (50mM Tris at ph 8.0, 120mM NaCl
More informationPlasmid DNA transfection of LN-229 human glioblastoma cells with the Biontex K2 Transfection System
Plasmid DNA transfection of human glioblastoma cells with the Biontex K2 Transfection System Stephanie Hehlgans and Franz Rödel, Department of Radiotherapy and Oncology, Goethe- University Frankfurt, Theodor-Stern-Kai
More informationab BrdU Immunohistochemistry Kit
ab125306 - BrdU Immunohistochemistry Kit Instructions for Use For the detection and localization of bromodeoxyuridine incorporated into newly synthesized DNA of actively proliferating cells. This product
More informationIgG TrueBlot Protocol for Mouse, Rabbit or Goatderived Antibodies - For Research Use Only
IgG TrueBlot Protocol for Mouse, Rabbit or Goatderived Antibodies - For Research Use Only Introduction The IgG TrueBlot for mouse, rabbit, or goat-derived antibodies represents unique series of respective
More informationUser Manual. OriCell TM Mesenchymal Stem Cell Osteogenic Differentiation Medium. Cat. No. GUXMX-90021
User Manual OriCell TM Mesenchymal Stem Cell Osteogenic Differentiation Cat. No. GUXMX-90021 PRODUCT DESCRIPTION: OriCell TM Mesenchymal Stem Cell Osteogenic Differentiation consists of optimized Mesenchymal
More informationMice TRAMP mice were maintained in a C57BL/6J background. Syngeneic UBI-GFP/BL6 mice were used for bone marrow engraftment. 2
Antibodies Chicken IgY polyclonal α-gfp antibodies were purchased from Abcam (Cambridge, MA) and were detected using α-chicken IgY-FITC or α-chicken-hrp (also purchased from Abcam). The CD31-PE, CD11b-PE,
More informationHuman skin punch biopsies were obtained under informed consent from normal healthy
SUPPLEMENTAL METHODS Acquisition of human skin specimens. Human skin punch biopsies were obtained under informed consent from normal healthy volunteers (n = 30) and psoriasis patients (n = 45) under a
More informationSupplementary Materials and Methods
Supplementary Materials and Methods Reagents Supplementary Material (ESI) for Lab on a Chip RPMI medium, FBS, HEPES buffer solution, sodium pyruvate, penicillin, and streptomycin were obtained from Biological
More informationTo determine MRK-003 IC50 values, cell lines were plated in triplicate in 96-well plates at 3 x
Supplementary methods: Cellular growth assay and cell cycle analysis To determine MRK-003 IC50 values, cell lines were plated in triplicate in 96-well plates at 3 x 10 3 cells/well (except for TALL1 and
More informationPlasmid DNA transfection of SW480 human colorectal cancer cells with the Biontex K2 Transfection System
Plasmid DNA transfection of human colorectal cancer cells with the Biontex K2 Transfection System Stephanie Hehlgans and Franz Rödel, Department of Radiotherapy and Oncology, Goethe- University Frankfurt,
More informationEffects of KAATSU Training on proliferation and differentiation of goat bone marrow mesenchymal stem cells
Chinese Journal of Laboratory Diagnosis Issued on 25 Aug 2016 Vol. 20, No. 8 P.1240 Effects of KAATSU Training on proliferation and differentiation of goat bone marrow mesenchymal stem cells YANG Yu-hui,
More information(Supplementary Methods online)
(Supplementary Methods online) Production and purification of either LC-antisense or control molecules Recombinant phagemids and the phagemid vector were transformed into XL-1 Blue competent bacterial
More informationMitoBiogenesis In-Cell ELISA Kit (Colorimetric)
PROTOCOL MitoBiogenesis In-Cell ELISA Kit (Colorimetric) DESCRIPTION 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 MS643 Rev.2 For identifying inhibitors and activators of mitochondrial biogenesis
More informationab BrdU Immunohistochemistry Kit
ab125306 - BrdU Immunohistochemistry Kit Instructions for Use For the detection and localization of bromodeoxyuridine incorporated into newly synthesized DNA of actively proliferating cells. This product
More informationSUPPLEMENTAL MATERIAL. Supplemental Methods:
SUPPLEMENTAL MATERIAL Supplemental Methods: Immunoprecipitation- As we described but with some modifications [22]. As part of another ongoing project, lysate from human umbilical vein endothelial cells
More informationab Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis
ab139418 Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis Instructions for Use To determine cell cycle status in tissue culture cell lines by measuring DNA content using a flow cytometer. This
More informationab Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis
ab139418 Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis Instructions for Use To determine cell cycle status in tissue culture cell lines by measuring DNA content using a flow cytometer. This
More informationPreparation of Mouse Bone Marrow Stromal Cells
Preparation of Mouse Bone Marrow Stromal Cells A single-step stem cell purification method using adhesion to cell culture plastic was employed as described in the Reference. Briefly, neonatal and adult
More informationElectronic Supplementary Information
Electronic Supplementary Information FRET-based probing to gain direct information on sirna sustainability in live cells: Asymmetric degradation of sirna strands Seonmi Shin, a Hyun-Mi Kwon, b Kyung-Sik
More informationA Cell-Surface-Anchored Ratiometric I-Motif Sensor for. Extracellular ph Detection
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 216 Electronic Supplementary Information (ESI) A Cell-Surface-Anchored Ratiometric I-Motif Sensor for
More informationTrkB knockdown cell lines (i.e., BBM1-KD and 361-KD cells) were prepared by
Supplemental Information Cell Culture TrkB knockdown cell lines (i.e., BBM1-KD and 361-KD cells) were prepared by transducing BBM1 or 361 cells with a lentivirus encoding shrna for TrkB. Transduction was
More informationGraphene oxide-enhanced cytoskeleton imaging and mitosis tracking
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2017 Supplementary information for Graphene oxide-enhanced cytoskeleton imaging and mitosis tracking
More informationChemical mixtures isolated from house dust disrupt thyroid receptor β (TRβ) signaling
SUPPORTING INFORMATION Chemical mixtures isolated from house dust disrupt thyroid receptor β (TRβ) signaling Erin M. Kollitz, Christopher D. Kassotis, Kate Hoffman, P. Lee Ferguson, Julie Ann Sosa, Heather
More informationAt E17.5, the embryos were rinsed in phosphate-buffered saline (PBS) and immersed in
Supplementary Materials and Methods Barrier function assays At E17.5, the embryos were rinsed in phosphate-buffered saline (PBS) and immersed in acidic X-gal mix (100 mm phosphate buffer at ph4.3, 3 mm
More informationMeCP2. MeCP2/α-tubulin. GFP mir1-1 mir132
Conservation Figure S1. Schematic showing 3 UTR (top; thick black line), mir132 MRE (arrow) and nucleotide sequence conservation (vertical black lines; http://genome.ucsc.edu). a GFP mir1-1 mir132 b GFP
More informationBioimaging of microrna-294 expression-dependent color change. in cells by a dual fluorophore-based molecular beacon
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2014 Electronic Supplementary Information for Chemical Communications Bioimaging of microrna-294 expression-dependent
More informationKnockdown of Arhgef-1 expression in VSMCs To knockdown rat VSMCs Arhgef-1 expression, three sets of sirna against rat
Supplemental Material Chemicals and Reagents PGE2 and sulprostone were respectively purchased from Cayman Chemical (Ann Arbor, MI) and BioMol Research Laboratories (Plymouth Meeting, PA). M&B28767 was
More informationFig. S1 TGF RI inhibitor SB effectively blocks phosphorylation of Smad2 induced by TGF. FET cells were treated with TGF in the presence of
Fig. S1 TGF RI inhibitor SB525334 effectively blocks phosphorylation of Smad2 induced by TGF. FET cells were treated with TGF in the presence of different concentrations of SB525334. Cells were lysed and
More informationOnline supplement to: Activation of the Alternative Complement Pathway in Vitreous is. Controlled by Genetics in Age-related Macular Degeneration
Online supplement to: Activation of the Alternative Complement Pathway in Vitreous is led by Genetics in Age-related Macular Degeneration Kelly M. Loyet, 1 Laura E. DeForge, 1 Kenneth J. Katschke, Jr.
More informationHigh throughput screening: Huh-7 cells were seeded into 96-well plate (2000
1 SUPPLEMENTARY INFORMATION METHODS 6 7 8 9 1 11 1 1 1 1 16 17 18 19 High throughput screening: Huh-7 cells were seeded into 96-well plate ( cells/well) and infected with MOI of DENV-. One hour post-infection
More informationAdeno-X Rapid Titer Kit User Manual
Adeno-X Rapid Titer Kit User Manual Cat. No. 631028 PT3651-1 (PR5X1070) Published 30 September 2005 Table of Contents I. Introduction & Protocol Overview 3 II. List of Components 5 III. Additional Materials
More informationNTM486-04, NTM174-04,
Transfection of transformed human trabecular meshwork TM5, and primary human NTM210-05, NTM486-04, NTM174-04, and NTM153-00 cells with Metafectene Easy Adnan Dibas1A,C, Ming Jiang1A,C, Thomas Yorio1A,C.
More informationElectromagnetical stimulation of human articular chondrocytes by. the Algonix device.
Electromagnetical stimulation of human articular chondrocytes by the Algonix device. Karsten Gavénis 1*, Stefan Andereya 1, Bernhard Schmidt-Rohlfing 2, Ralf Mueller-Rath 1, Jiri Silny 3, Ulrich Schneider
More informationFor identifying inhibitors and activators of mitochondrial biogenesis in adherent cultured cells.
ab110216 MitoBiogenesis TM In-Cell ELISA Kit (IR) Instructions for Use For identifying inhibitors and activators of mitochondrial biogenesis in adherent cultured cells. This product is for research use
More informationSupporting Information
Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 2015 Supporting Information Materials BSA (bovine serum albumin, 99%), fluorescein isothiocyanate
More informationApplication Note 18 RNA/DNA/Protein Sample Preparation METHODS AND MATERIALS INTRODUCTION
Application Note 18 /DNA/Protein Sample Preparation Sequential Purification of, DNA and Protein from a Single Sample using 's /DNA/Protein Purification Kit and Comparison to a Market B. Lam, PhD 1, C.
More information3T3-L1 Differentiation Protocol
3T3-L1 Differentiation Protocol Written by Eisuke Kato on 2013/10/09 MATERIALS Dulbecco's Modified Eagles Medium (D-MEM) (High Glucose) with L-Glutamine and Phenol Red High glucose (Wako Chem 044-29765,
More informationSingle cell imaging of Bruton's Tyrosine Kinase using an irreversible inhibitor
SUPPLEMENTARY INFORMATION Single cell imaging of Bruton's Tyrosine Kinase using an irreversible inhibitor Anna Turetsky 1,a, Eunha Kim 1,a, Rainer H. Kohler 1, Miles A. Miller 1, Ralph Weissleder 1,2,
More informationAdeno-X Rapid Titer Kit
User Manual Adeno-X Rapid Titer Kit User Manual United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116 Clontech Laboratories, Inc. A Takara Bio
More informationUsing the xcelligence RTCA SP Instrument to Perform GPCR Assays
xcelligence Real-Time Cell Analysis GPCR Assay Protocol Using the xcelligence RTCA SP Instrument to Perform GPCR Assays Overview The xcelligence RTCA SP Instrument The xcelligence Real-Time Cell Analyzer
More informationSANTA CRUZ BIOTECHNOLOGY, INC.
TECHNICAL SERVICE GUIDE: Western Blotting 2. What size bands were expected and what size bands were detected? 3. Was the blot blank or was a dark background or non-specific bands seen? 4. Did this same
More informationSupplementary Figure 1 A green: cytokeratin 8
Supplementary Figure 1 A green: cytokeratin 8 green: α-sma red: α-sma blue: DAPI blue: DAPI Panc-1 Panc-1 Panc-1+hPSC Panc-1+hPSC monoculture coculture B Suppl. Figure 1: A, Immunofluorescence staining
More informationBrdU IHC Kit. For the detection and localization of bromodeoxyuridine incorporated into newly synthesized DNA of actively proliferating cells
K-ASSAY BrdU IHC Kit For the detection and localization of bromodeoxyuridine incorporated into newly synthesized DNA of actively proliferating cells Cat. No. KT-077 For Research Use Only. Not for Use in
More informationTo determine the effect of N1IC in the susceptibility of T cells to the tolerogenic effect of tumorassociated
Supplementary Methods Tolerogenic effect of MDSC To determine the effect of N1IC in the susceptibility of T cells to the tolerogenic effect of tumorassociated MDSC in vivo, we used a model described previously
More informationJung-Nam Cho, Jee-Youn Ryu, Young-Min Jeong, Jihye Park, Ji-Joon Song, Richard M. Amasino, Bosl Noh, and Yoo-Sun Noh
Developmental Cell, Volume 22 Supplemental Information Control of Seed Germination by Light-Induced Histone Arginine Demethylation Activity Jung-Nam Cho, Jee-Youn Ryu, Young-Min Jeong, Jihye Park, Ji-Joon
More informationSupplementary Information Alternative splicing of CD44 mrna by ESRP1 enhances lung colonization of metastatic cancer cell
Supplementary Information Alternative splicing of CD44 mrna by ESRP1 enhances lung colonization of metastatic cancer cell Supplementary Figures S1-S3 Supplementary Methods Supplementary Figure S1. Identification
More informationSupporting Information. A Fluorogenic Resveratrol-Confined Graphene Oxide For Economic and Rapid. Detection Of Alzheimer's Disease
Supporting Information A Fluorogenic Resveratrol-Confined Graphene Oxide For Economic and Rapid Detection Of Alzheimer's Disease Xiao-Peng He, 1 Qiong Deng, 1 Liang Cai, 1 Chang-Zheng Wang, 1,2 Yi Zang,*,2
More informationEstablishing sirna assays in primary human peripheral blood lymphocytes
page 1 of 7 Establishing sirna assays in primary human peripheral blood lymphocytes This protocol is designed to establish sirna assays in primary human peripheral blood lymphocytes using the Nucleofector
More informationebioscience BrdU Kit for IHC/ICC Colorimetric Catalog Number: RUO: For Research Use Only. Not for use in diagnostic procedures.
Page 1 of 1 ebioscience BrdU Kit for IHC/ICC Colorimetric Catalog Number: 8800-6599 RUO: For Research Use Only. Not for use in diagnostic procedures. Product Information Contents: ebioscience BrdU Kit
More information1 Electronic Supplementary information. 2 Co-assembling FRET nanomedicine with self-indicating drug
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2018 1 Electronic Supplementary information 2 Co-assembling FRET nanomedicine with self-indicating drug
More informationPromotion of HDF Cell Attachment and Proliferation
Promotion of HDF Cell Attachment and Proliferation Objectives To qualitatively assess the effect of fibronectin (Fn) on HDF cell attachment Fn Attachment Assay To observe HDF cell proliferation and position
More informationFigure S Relative MUC4 transcript level* CD18/HPAF CD18/HPAF-Scr CD18/HPAF-siMUC4
Figure S1 Relative MUC4 transcript level* 1.4 1.2 1 0.8 0.6 0.4 0.2 0 CD18/HPAF CD18/HPAF-Scr CD18/HPAF-siMUC4 Figure S2 * * CD18/HPAF-Scr CD18/HPAF-siMUC4 CD18/HPAF-Scr CD18/HPAF-siMUC4 Figure S3 CD18/HPAF-Scr
More informationPE11, a PE/PPE family protein of Mycobacterium tuberculosis is involved in cell wall remodeling and virulence
PE11, a PE/PPE family protein of Mycobacterium tuberculosis is involved in cell wall remodeling and virulence Parul Singh 1,2, Rameshwaram Nagender Rao 1, Jala Ram Chandra Reddy 3, R.B.N. Prasad 3, Sandeep
More informationTransfection of CRISPR/Cas9 Nuclease NLS ribonucleoprotein (RNP) into adherent mammalian cells using Lipofectamine RNAiMAX
Transfection of CRISPR/Cas9 Nuclease NLS ribonucleoprotein (RNP) into adherent mammalian cells using Lipofectamine RNAiMAX INTRODUCTION The CRISPR/Cas genome editing system consists of a single guide RNA
More informationSupplementary Material
Supplementary Material Supplementary Methods Cell synchronization. For synchronized cell growth, thymidine was added to 30% confluent U2OS cells to a final concentration of 2.5mM. Cells were incubated
More informationLumiPico ECL Kit. ShineGene. User Manual. For Western Blot. Cat.Nos.ZK00901(12.5ml 2) ZK00902(50.0ml 2) LumiPico ECL Kits User Manual
LumiPico ECL Kits User Manual ShineGene LumiPico ECL Kit For Western Blot User Manual Cat.Nos.ZK00901(12.5ml 2) ZK00902(50.0ml 2) USD44.46 USD175.20 Published 24 Feb 2007 ShineGene LumiPico ECL Kits User
More informationElectronic Supplementary Information (ESI)
Electronic Supplementary Material (ESI) for Chemical Science. This journal is The Royal Society of Chemistry 2014 Electronic Supplementary Information (ESI) Gold(III) complexes inhibit growth of cisplatin-resistant
More informationConventional isoelectric focusing with the Agilent 3100 OFFGEL Fractionator
Conventional isoelectric focusing with the Agilent 3100 OFFGEL Fractionator Technical Overview Introduction This Technical Overview demonstrates the ability of the Agilent 3100 OFFGEL Fractionator to fractionate
More informationSupplemental Figure 1 (Figure S1), related to Figure 1 Figure S1 provides evidence to demonstrate Nfatc1Cre is a mouse line that directed gene
Developmental Cell, Volume 25 Supplemental Information Brg1 Governs a Positive Feedback Circuit in the Hair Follicle for Tissue Regeneration and Repair Yiqin Xiong, Wei Li, Ching Shang, Richard M. Chen,
More informationSupporting Information
Supporting Information Self-Powered Electrical Stimulation for Enhancing Neural Differentiation of Mesenchymal Stem Cells on Graphene-Poly(3,4-ethylenedioxythiophene) Hybrid Microfibers Weibo Guo, 1,2
More informationSupplemental Table 1: Sequences of real time PCR primers. Primers were intronspanning
Symbol Accession Number Sense-primer (5-3 ) Antisense-primer (5-3 ) T a C ACTB NM_001101.3 CCAGAGGCGTACAGGGATAG CCAACCGCGAGAAGATGA 57 HSD3B2 NM_000198.3 CTTGGACAAGGCCTTCAGAC TCAAGTACAGTCAGCTTGGTCCT 60
More informationSupplementary Table 1. Primers used to construct full-length or various truncated mutants of ISG12b2.
Supplementary Table 1. Primers used to construct full-length or various truncated mutants of ISG12b2. Construct name ISG12b2 (No tag) HA-ISG12b2 (N-HA) ISG12b2-HA (C-HA; FL-HA) 94-283-HA (FL-GFP) 93-GFP
More informationBrdU Immunohistochemistry Kit Instruction Manual
BrdU Immunohistochemistry Kit Instruction Manual Features Easy to use system Reagents titered for success Proven protocol Ordering Information Catalog Number X1545K Size 50 Slides Format Immunohistochemistry
More informationFlow cytometry Stained cells were analyzed and sorted by SORP FACS Aria (BD Biosciences).
Mice C57BL/6-Ly5.1 or -Ly5.2 congenic mice were used for LSK transduction and competitive repopulation assays. Animal care was in accordance with the guidelines of Keio University for animal and recombinant
More informationData Sheet. TCR activator / PD-L1 - CHO Recombinant Cell line Cat. #: 60536
Data Sheet TCR activator / PD-L1 - CHO Recombinant Cell line Cat. #: 60536 Product Description Recombinant CHO-K1 cells constitutively expressing human PD-L1 (Programmed Cell Death 1 Ligand 1, CD274, B7
More informationSupplementary Online Material
Material and Methods Supplementary Online Material Reagents and antibodies Wortmannin, JNK inhibitor II (Anthra[1,9-cd]pyrazol-6(2H)-one 1,9-pyrazoloanthrone), SB 2358, and PD 9859 were purchased from
More informationBmDCs were generated as described by a modified protocol of Inaba et al S1. Briefly, bone
Generation and culture of bone marrow-derived dendritic cells (bmdcs) BmDCs were generated as described by a modified protocol of Inaba et al S1. Briefly, bone marrow cells from murine tibias and femurs
More informationModeling Cardiac Hypertrophy: Endothelin-1 Induction with qrt-pcr Analysis
icell Cardiomyocytes Application Protocol Modeling Cardiac Hypertrophy: Endothelin-1 Induction with qrt-pcr Analysis Introduction Cardiac hypertrophy is characterized by several different cellular changes,
More information