Explants of the periodontitis-affected and healthy periodontal tissues were collected from patients undergoing extraction and cultured. The middle part of the root was gently scraped to collect periodontal ligament (PDL) tissue. PDL tissues were cut into 1 mm 3 cubes and transferred to a 6-well plate (Costar, Cambridge, USA) containing Minimum Essential Medium (MEM, Sigma-Aldrich, St. Louis, USA) supplemented with 0.292 mg/mL glutamine (Sigma-Aldrich, St. Louis, USA), 100 µM ascorbic acid (Sigma-Aldrich, St. Louis, USA), 100 U/mL penicillinstreptomycin (Sigma-Aldrich, St. Louis, USA), and 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, USA). Cells were grown at 37°C in a 5% CO 2 incubator. The isolation of PDLSCs was performed using the same method described in a previous study 11 . To separate PDLSCs, all cells were incubated with mouse anti-human monoclonal stem cell marker STRO-1 (1:100; R&D Systems, Minneapolis, USA), and the STRO-1 + stem cells were isolated using immunomagnetic beads 11 .
Stro 1
Manufactured by R&D Systems
Sourced in United States
Stro-1 is a cell surface marker that is expressed on human mesenchymal stem cells (MSCs). It is commonly used for the identification and isolation of MSCs from various tissue sources.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Cd146, by Abcam (2 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Avantor (1 mentions)
Bz x700, by Keyence (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Cd105, by R&D Systems (1 mentions)
Cd106, by R&D Systems (1 mentions)
Canto 2, by BD (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
Anti cd34 fitc, by BioLegend (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Cd45 fitc, by BioLegend (1 mentions)
Cd73 fitc, by BioLegend (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
11 protocols using stro 1
1
Isolation and Cultivation of PDLSCs
2
Immunophenotyping of Stem Cell Cultures
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In order to confirm the identity of cells isolated and cultured by the aforementioned method, immunofluorescence using a panel of cell markers for SMSCs was performed. Positive and negative markers were selected based on a literature review 18 . Cells were plated in four‐chamber slides and allowed to achieve 60% confluence. The cells were then fixed, permeabilized, and blocked with 4% paraformaldehyde (VWR, Radnor, PA), 0.1% Triton X‐100 (Sigma‐Aldrich, St. Louis, MO), and 1% bovine serum albumin (Sigma‐Aldrich). Cells were incubated overnight with primary antibodies against CD31 (Abcam, Cambridge, UK), CD44, CD45, CD90, CD105, CD106, and STRO‐1 (R&D Systems, Minneapolis, MN), followed by incubation with a secondary antibody (Alexa Fluor 488, ThermoFisher Scientific, Waltham, MA). In order to more specifically confirm the identity of these cells, costaining was performed using a similar protocol with antibodies against CD90 (R&D Systems) and CD44 (Abcam). Secondary antibodies for costaining were Alexa Fluor 488 and 594, respectively (ThermoFisher Scientific). All images were obtained using a Keyence BZ‐X700 (Keyence, Osaka, Japan).
3
Mesenchymal Stem Cell Phenotyping by FACS
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Cells grown to an optimal confluence of 70% were used for FACS analysis. Single cell suspension was prepared by dissociating the cells using 0.05% Trypsin and triturating in PFN (PBS + 5%FBS + 0.1%Sodium Azide). The cells were fixed with 4% paraformaldehyde in PFN over ice for 1 hour. The fixed cells were incubated with two primary antibodies per experiment (CD29[Abcam, 1:100]/CD146[Abcam, 1:50 and CD44[Abcam, 1:50]/Stro1[R&D Systems, 1:50]) at 4 °C overnight. Subsequently, the cells were incubated with fluorescent secondary antibodies over ice for 1 hour and analysed by flow-cytometer (Canto II, BD Biosciences). FlowJo software was used for gating and further analysis.
4
Expansion and characterization of human BMD-MSCs
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The AGS cell line (human gastric adenocarcinoma cell line) was purchased from Iran Pasteur Institute (Tehran, Iran) and were cultured in RPMI 1640 supplemented with 10% FBS (Gibco, Manchester, UK) at 37°C in a humid incubator with 5% CO2. Human BMD-MSCs were expanded in vitro from the bone marrow of healthy donors after informed consent was obtained. Mononuclear cells (MNCs) were isolated by gradient centrifugation at 2,500 rpm for 30 min on Ficoll-Paque™ Plus (Amersham Pharmacia Biotech, Uppsala, Sweden). Then, plated at a concentration of 20-30 × 106 cells/cm2 in Dulbecco's Modified Eagle Medium (DMEM) containing 20% (v/v) of FBS. Then, nonadherent cells were removed 2 days later and a fresh medium was added. BMD-MSCs were trypsinized when the cultures reached 80-100% confluence and subcultured. The purity of MSC suspensions was assessed by flow cytometry using the following monoclonal antibodies: anti-CD34-FITC, CD45-FITC, CD73-FITC (all from Biolegend, Ankara, Turkey), and Stro-1 (R&D, Istanbul, Turkey).
5
Isolation of Myometrial and Fibroid Stem Cells
6
Phenotypic Analysis of DPSCs
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For identification of DPSCs phenotype, 3 × 10E5 DPSCs (passage 3) were incubated with PE- or FITC-conjugated monoclonal antibodies against human CD29, CD44, CD73, CD90, CD105, CD34, CD45 (eBioscience, CA), and Stro-1 (R&D Systems, MN) (sFig. 1 ), and flow cytometric analysis were performed using a Beckman CytoFLEX S flow cytometer (Beckman Coulter, USA).
7
Flow Cytometric Analysis of Stem Cell Markers
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All steps were performed at 22 °C unless otherwise specified. Cells were dissociated from cultures by brief treatment with TrypLE Express (37 °C) and centrifuged at 250 g for 5 min. Cells were fixed using 4 % paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min and then washed with PBS. Cells were resuspended in blocking buffer comprising PBS with 3 % v/v normal goat serum (NGS; Millipore) and 0.01 % v/v Triton X-100 (Sigma-Aldrich) for 30 min. After centrifugation, cells were incubated with selected primary antibody in blocking buffer for 2 h. Thereafter, cell pellets were resuspended in appropriate secondary antibody in blocking buffer for 1 h. Cells were subjected to flow cytometric analysis with the use of BD Canto II analyzer. For each marker, at least 10,000 cells were analyzed.
Primary antibodies used were: CD90 (mouse anti-rat/human, 1:200; BD Bioscience), CD73 (mouse anti-rat/human, 1:200; BD Bioscience), STRO-1 (mouse anti-rat/human, 1:50; R&D Systems), CD45 (mouse anti-rat/human, 1:200; BD Bioscience), and nestin (mouse anti-rat/human, 1:200; BD Bioscience). Mouse isotype control (1:200; Life Technologies) was used in the control experiments. Secondary antibody used was donkey anti-mouse Alexa 488 (1:500; Life Technologies).
Primary antibodies used were: CD90 (mouse anti-rat/human, 1:200; BD Bioscience), CD73 (mouse anti-rat/human, 1:200; BD Bioscience), STRO-1 (mouse anti-rat/human, 1:50; R&D Systems), CD45 (mouse anti-rat/human, 1:200; BD Bioscience), and nestin (mouse anti-rat/human, 1:200; BD Bioscience). Mouse isotype control (1:200; Life Technologies) was used in the control experiments. Secondary antibody used was donkey anti-mouse Alexa 488 (1:500; Life Technologies).
8
Characterization of Stem Cell Markers
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TSPC at passage 6 were plated and cultured on 20 μg/ml collagen 1-coated glass slides (BD Bioscience, USA) for 48 h. Then, the cells were fixed with 4% paraformaldehyde (Merck, Germany), permeabilized with Triton X100 (Sigma-Aldrich) and blocked with 3% BSA (PAA, USA). Primary antibodies against CD146 (Millipore, USA), Nestin (Proteintech, USA) and STRO-1 (R&D Systems, USA) were applied overnight at 4°C. Next, secondary Alexa Flour 488-conjugated antibodies and DAPI were used (all Life technologies, USA). As negative control were used cell-seeded slides which were incubated only with secondary Alexa Flour 488-conjugated antibodies and DAPI. Photomicrographs were taken with Axiocam MRm camera on Axioskope 2 microscope (Carl Zeiss, Germany). Staining procedures were reproduced at least twice.
9
Characterization of Bone Marrow-Derived Mesenchymal Stem Cells Exposed to Helicobacter pylori
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BMD-MSCs were cocultured with H. pylori for 24 h, harvested, and washed with FCM buffer (PBS containing 1% BSA) three times. Cells were stained as described previously.[23 (link)] Briefly, cells were incubated with 10 μg/mL of mouse anti-human CXCR4 (Santa Cruz, Dallas, TX) or isotype antibody (Dako, Glostrup, Denmark) for 45 min on ice, then washed and incubated with goat anti-mouse FITC conjugated secondary antibody (Dako) for 30 min. Cells then were washed three times, fixed in 1% paraformaldehyde, and subjected to FACS analysis (FACS Calibur, Beckman Dickinson, San Jose, CA). The purity of BMD-MSCs was determined using stained anti-CD45-FITC, CD73-FITC, CD34-FITC, and Stro-1. Cells were incubated with 10 μL of either anti-CD45-FITC, CD73-FITC, or CD34 FITC for 30 min at room temperature, washed three times with FCM buffer, and subjected to FACS analysis. To stain BMD-MSCs for Stro-1 (R&D, Minneapolis, MN) cells were incubated with anti-Stro-1 monoclonal antibody for 45 min on ice, then washed and incubated with secondary antibody as mentioned above. FACS data were analyzed by FCS Express software (De Novo Software, Los Angeles, CA).
10
Immunohistochemical Characterization of Chondrogenic Cells
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Indirect immunoperoxidase labeling was carried out using human specific antibodies against collagens type I (COL-1, Sigma Aldrich, UK), II (Abcam, UK), X (Gift from Klaus von der Mark), and aggrecan (5C5, Enzo Life Sciences, UK). Fluorescence labeling was used to detect Stro-1 (R&D Systems, UK). Primary antibodies were diluted in 0.1 M PBS containing 0.01% Tween 20 (PBS-T) at a concentration of 10 µg mL−1. Appropriate antigen retrieval techniques were necessary to expose the collagen, aggrecan, and Stro-1 epitopes. For collagen types I and II, pellets were subjected to a chondroitinase (0.25 U mL−1; Sigma Aldrich, UK) and hyaluronidase (2 U mL−1; Sigma Aldrich, UK) pretreatment for 1.5 hours at 37°C. For collagen type X, pellets were first subjected to a proteinase K (2.0 µg mL−1; Sigma Aldrich, UK) digest for 1 hour at 37°C, and subsequently a chondroitinase (0.25 U mL−1) and hyaluronidase (2 U mL−1) treatment for 30 minutes at 37°C. For aggrecan (5C5), pellets were subjected to a hyaluronidase (2 U mL−1) pretreatment for 1.5 hours at 37°C. Pellets from a minimum of three different clonal cell lines were labeled for each antibody. For Stro-1, pellets were pretreated with 0.3% triton X (in PBS) for 30 minutes at room temperature.
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