Flow cytometric analysis was performed to evaluate the surface markers of hBMSCs. The hBMSCs cultured with PL in CPC were suspended with PBS containing 3% FCS. They were incubated with either a mouse monoclonal antibody against human CD19 (R&D Systems; dilution, 1 : 100), CD44 (R&D Systems; 1 : 100), CD45 (R&D Systems; 1 : 100), CD90 (R&D Systems; 1 : 100), CD105 (R&D Systems; 1 : 100), CD106 (R&D Systems; 1 : 100), CD146 (R&D Systems; 1 : 100), CD166 (R&D Systems; 1 : 100), or each mouse isotypic control for 30 min on ice. Cell suspensions were then incubated with Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes, Thermo Fisher Scientific; 1 : 200) for 30 min on ice. Flow cytometric analysis was performed after two washes using a cytometer (Attune® Acoustic Focusing Cytometer, Applied Biosystems, Thermo Fisher Scientific). Live events (10,000) were acquired for analysis.
Cd106
Manufactured by R&D Systems
Sourced in United Kingdom, China
CD106 is a laboratory reagent that can be used for research purposes. It is a transmembrane glycoprotein that functions as a cell adhesion molecule. CD106 plays a role in the regulation of leukocyte-endothelial cell adhesion and migration.
Automatically generated - may contain errors
Lab products found in correlation
Cd105, by R&D Systems (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Attune acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Cd146, by R&D Systems (1 mentions)
Cd166, by R&D Systems (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), 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)
Stro 1, by R&D Systems (1 mentions)
Cd105, by Thermo Fisher Scientific (1 mentions)
Sca 1, by Thermo Fisher Scientific (1 mentions)
Cd11b, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
6 protocols using cd106
1
Characterizing hBMSC Surface Markers
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
Isolation and Characterization of Mouse MSCs
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2M WT and CKIP-1 KO mice were executed by cervical dislocation. The tibias and femurs were immediately dissected from the attached muscles and tissues using aseptic techniques. The ends of the bones were removed, marrow plugs were flushed by repeated pipetting, and the cells were forcefully passed through a 19-gauge needle to obtain a single-cell suspension. The cells were cultured in a growth medium containing α-minimum eagle's medium (α-MEM; Invitrogen, Beijing, China) made of 10% fetal bovine serum and 1% penicillin. The cell suspensions were seeded in 10-cm tissue culture dishes and cultured in growth medium in a humidified atmosphere of 5% CO2 at 37°C. The culture media were changed every 2 to 3 days to remove non-adherent cells, and adherent cells were grown until they were confluent. At last, MSCs were sub-cultured after digestion with 0.25% trypsin and 1 mol/L ethylenediamine tetraacetic acid (EDTA) (Yaji Bio-technology Co., Ltd., Shanghai, China). A fluorescence-activated cell sorting analysis was used to analyze the expression of CD44, CD90, CD106, CD34, and CD45 (R&D Systems, Fushen Bio-technology Co., Ltd., Shanghai, China) according to the manufacturer's protocols.
4
Characterization of Isolated Alveolar Mesenchymal Stem Cells
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Flow cytometry was used to examine the purity of isolated AFMSCs by detection CD44, stem cell antigen 1 (Sca-1), CD105 (eBioscience), CD34, CD90 (BD Biosciences), CD29, CD11b, CD73, CD106, and CD45 (R&D Systems) expression on the cell surface as described in Peng et al. [29 (link)] (Additional file 2 : Figure S1E). To confirm the percentage of differentiated AFMSCs towards LEPLCs, we detect the presence of TTF-1, SP-C, AQP-5, and CCSP in cells according to the manufacturer’s instructions. Briefly, detached cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 for 15 min, blocked with 4% BSA in PBS for 1 h, and then incubated overnight at 4 °C with the following intracellular marker antibodies at appropriate dilutions: TTF-1 (Abcam, 1:250 dilution), SP-C, AQP-5, and CCSP (Millipore, all at 1:250 dilutions). The cells were then incubated with an appropriate Alexa Fluor® 546 dye-conjugated secondary antibody (1:200) at room temperature for 2 h. After rinsing the cells twice, fluorescence was detected and analyzed using flow cytometry (FACSCalibur).
5
Isolation and Characterization of hBMMSCs
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Primary human BMMSCs were isolated as previously described [16 (link)]. The sample collection was conformed to protocol approved by the ethical authorities at the 3201 Hospital Affiliated to Xi'an Jiaotong University. The experiment was conducted with the donors' or subjects' understanding and consent, as well as a statement that the responsible Ethical Committee has approved the experiments.
First, healthy bone marrow samples were collected from 5 donors, aged from 18 to 30 years, undergoing the alveolar bone cleft repair by autoilium transplantation. 1 mL bone marrow aspirates were seeded into a 10 cm culture dish and added 20 mL α-MEM (Gibco BRL, Gaithersburg, MD) supplemented with 15% FBS (Gibco, BRL), then incubated in 5% CO2, 37°C. Cells from 3–5 passages were used in the experiments.
Second, fluorescence-activated cell sorting (FACS) analysis was used for analyzing CD45, CD34, CD14, CD29, CD44, CD90, and CD106 (R&D Systems, Inc.). Multiple colony-derived hMSCs at 2–4 passages were used in our experiments.
First, healthy bone marrow samples were collected from 5 donors, aged from 18 to 30 years, undergoing the alveolar bone cleft repair by autoilium transplantation. 1 mL bone marrow aspirates were seeded into a 10 cm culture dish and added 20 mL α-MEM (Gibco BRL, Gaithersburg, MD) supplemented with 15% FBS (Gibco, BRL), then incubated in 5% CO2, 37°C. Cells from 3–5 passages were used in the experiments.
Second, fluorescence-activated cell sorting (FACS) analysis was used for analyzing CD45, CD34, CD14, CD29, CD44, CD90, and CD106 (R&D Systems, Inc.). Multiple colony-derived hMSCs at 2–4 passages were used in our experiments.
6
Characterization of rBMSCs by Flow Cytometry
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Passage 3 rBMSCs were collected after 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) treatment and washed with PBS for three times. The cell concentration was adjusted to 1.0x106 cells/ml, and incubated for 25-30 min with antibodies against the following cell surface antigens: CD29 (dilution, 1:50; cat. no. AF2405; R&D Systems), CD106 (dilution, 1:40; cat. no. ab134047; Abcam), CD34 (dilution, 1:50; cat. no. ab81289; Abcam) and CD45 (dilution, 1:50; cat. no. ab10558; Abcam). Cells were washed three times and incubated with FITC-conjugated anti-goat (dilution, 1:200; cat. no. ab6881; Abcam) or FITC-conjugated anti-rabbit (dilution, 1:200; cat. no. ab6717; Abcam) secondary antibodies. Flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter, Inc.), and the data were analyzed with FlowJo software (version 10.4.2; FlowJo LLC).
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