Medaka mesonephros and adult C57Bl/6 mouse kidneys were mechanically dissociated to a single cell suspension. Whole kidneys and KKPS5 cells were stained for CD133-PE, CD34-PE, CD105-PE, CD90.2-FITC (eBiosciences, Inc., San Diego, CA), Sca-I–Pacific Blue, c-kit-PE-Cy7 (BioLegend, San Diego, CA), CD31-FITC, CD24-FITC, CD106-FITC, FLT-3-PE, CD9-Biotin, and Streptavidin-APC-Cy7 (BD Biosciences), and analyzed by flow cytometry using an LSRII (BD Biosciences) with FACSDiva (BD Biosciences) software version 4.1. Appropriate isotype controls (BD Biosciences, eBiosciences, Inc., BioLegend) were used for analyses. Doublet exclusion was used to ensure analyses of single cells prior to forward/side scatter gating. Data were analyzed using FlowJo (Tree Star, Inc., Ashland, OR) software version 10.
Cd106 fitc
Manufactured by BD
Sourced in United States
CD106-FITC is a fluorescently labeled antibody that binds to the cell surface antigen CD106, also known as VCAM-1. It is used in flow cytometry applications to identify and quantify cells expressing CD106.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 pe, by BD (6 mentions)
Cd73 pe, by BD (6 mentions)
Cd44 pe, by BD (4 mentions)
Cd29 pe, by BD (4 mentions)
Cd45 pe, by BD (4 mentions)
Cd45 fitc, by BD (4 mentions)
Cd31 fitc, by BD (3 mentions)
Cd29 apc, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Cd49e pe, by BD (3 mentions)
Cd54 pe, by BD (3 mentions)
Cd14 alexa 700, by BD (2 mentions)
Cd90 pe cy7, by BD (2 mentions)
Cd90 pe, by BD (2 mentions)
Cd44 fitc, by BD (2 mentions)
Cd90 fitc, by BD (2 mentions)
Cd31 pe, by BD (2 mentions)
Cd14 fitc, by BD (2 mentions)
Cd166 pe, by BD (2 mentions)
Cd34 fitc, by BD (2 mentions)
13 protocols using cd106 fitc
1
Characterization of Murine Kidney Cell Populations
2
Immunophenotyping of hBM-MSC with MFNP
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The immunophenotyping of hBM-MSC unlabeled and labeled with 50 µg Fe/mL of MFNP during 24 h (the maximum incubation time analyzed in this study) was performed using the following antibodies: CD19-FITC (clone:4G7) and CD34-PE (clone: 8G12) from BD Biosciences, San Jose, CA, USA; CD14-Alexa 700 (clone: M5E2), CD29-APC (clone: MAR04), CD35-FITC (clone: E11), CD44-PerCP-Cy5.5 (clone: G44-26), CD73-PE (clone: AD2), CD90-PE-Cy7 (clone: 5E10), HLA-DR-APC-H7 (clone: G46-6), CD106-FITC (clone: 51-10C9) all from BD Pharmingen, San Diego, CA, USA; CD31-V450 (clone: WM59), CD45-V500 (clone: H130), CD105-PE-CF546 (clone: 266) all from BD Horizon, San Jose, CA, USA, unstained samples and fluorescence minus one (FMO) was used as a fluorescence background control. Briefly, 1 × 105 cells were incubated in the dark/room temperature for 30 min in the presence of antibodies at concentrations recommended by the manufacturers. Cells were then washed with a buffered solution, and at least 10,000 events were acquired using FACS LSRII FORTESSA equipment (BD Biosciences). Data analyses were performed using FlowJoTM software (BD Biosciences), and results were presented as graphics where the FMO is overlaid with the stained sample for each antibody/marker used.
3
Phenotypic Characterization of Adherent AFCs
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Adherent AFCs were separated by trypsin treatment and fixed in 75% ethanol overnight at 4°C. The cells were filtered through a 40 μm mesh and resuspended in fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) and 0.1% sodium azide]. Directly-conjugated isotype control antibodies, IgG-fluorescein isothiocyanate (FITC) and IgG-phycoerythrin (PE; BD Pharmingen, San Diego, CA, USA), were used as controls to identify the background cells. A total of ~5×105 cells were incubated at 4°C for 40 min with each of the following FITC- or PE-conjugated antibodies (BD Pharmingen): CD133-PE, CD117-PE, CD34-PE, CD105-FITC, CD106-FITC, CD29-PE, CD44-FITC, CD147-FITC and CD90-PE. The cells were subsequently washed in FACS buffer. The antibody-labeled cells were analyzed using a BD FACSCalibur instrument (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed using FlowJo version 7.2.5 software (TreeStar, Inc., Ashland, OR, USA). The cell count experiments for the flow cytometry assays were performed in triplicate.
4
Characterization of ADSC Surface Markers
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Cell surface marker expression was examined as follows: Fluorochrome-conjugated anti-human CD14-FITC, CD31-FITC, CD34-PE, CD44-PE, CD29-APC, CD73-PE, and CD90-FITC antibodies and Fluorochrome-conjugated anti-mouse CD29-FITC, CD31-FITC, CD44-PE, CD45-PE, and CD106-FITC antibodies were purchased from BD Pharmingen (San Diego, CA, USA) and used in accordance with the instructions of the manufacturer. Non-specific staining was controlled by the use of isotype-matched antibodies. ADSC suspensions were incubated with the primary antibodies (1:50) for 30 min at room temperature. After incubation, the cells were washed twice with PBS and analyzed using a flow cytometer (BD FACSAria™ III system; BD Pharmingen).
5
Cell Surface Marker Expression Profiling
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We analyzed cell surface expression with a pre-defined set of protein markers. These
assays were performed using commercially available monoclonal antibodies, following the
manufacturers’ instructions. Briefly, the cells at third passage were harvested by a
treatment with 0.25% Tryple Express (Gibco-Invitrogen, Carlsbad, CA, USA), washed with PBS
(pH = 7.4) and stained with the selected monoclonal antibodies and incubated in the dark
for 30 min at 4°C. Cells were then washed and fixed with 1% paraformaldehyde. The
following human antibodies were used: CD14-FITC (clone: M5E2; BD Pharmingen, San Diego,
CA, USA), CD29-PE (clone: MAR4; BD Pharmingen), CD31-PE (clone: WM59; BD Pharmingen),
CD34-PE (clone: 581; BD Pharmingen), CD44-PE (clone: 515; BD Pharmingen), CD45-PerCPCy5
(clone: 2D1; BD Biosciences, San Jose, CA, USA), CD73-PE (clone: AD2; BD Pharmingen),
CD90-APC (clone: 5E10; BD Pharmingen), CD106-FITC (clone: 51-10C9; BD Pharmingen),
CD166-PE (clone: 3A6; BD Pharmingen), HLA-DR-PerCP-Cy5 (clone: L243; BD Biosciences), and
CD105-PE (clone: 8E11; Chemicon, Temecula, CA, USA). Cells were analyzed using FACSARIA
flow cytometry equipment (BD Biosciences) and data analyses were performed using FACSDIVA
software (BD Biosciences) or Flow Jo Software (TreeStar, Ashland, OR, USA).
assays were performed using commercially available monoclonal antibodies, following the
manufacturers’ instructions. Briefly, the cells at third passage were harvested by a
treatment with 0.25% Tryple Express (Gibco-Invitrogen, Carlsbad, CA, USA), washed with PBS
(pH = 7.4) and stained with the selected monoclonal antibodies and incubated in the dark
for 30 min at 4°C. Cells were then washed and fixed with 1% paraformaldehyde. The
following human antibodies were used: CD14-FITC (clone: M5E2; BD Pharmingen, San Diego,
CA, USA), CD29-PE (clone: MAR4; BD Pharmingen), CD31-PE (clone: WM59; BD Pharmingen),
CD34-PE (clone: 581; BD Pharmingen), CD44-PE (clone: 515; BD Pharmingen), CD45-PerCPCy5
(clone: 2D1; BD Biosciences, San Jose, CA, USA), CD73-PE (clone: AD2; BD Pharmingen),
CD90-APC (clone: 5E10; BD Pharmingen), CD106-FITC (clone: 51-10C9; BD Pharmingen),
CD166-PE (clone: 3A6; BD Pharmingen), HLA-DR-PerCP-Cy5 (clone: L243; BD Biosciences), and
CD105-PE (clone: 8E11; Chemicon, Temecula, CA, USA). Cells were analyzed using FACSARIA
flow cytometry equipment (BD Biosciences) and data analyses were performed using FACSDIVA
software (BD Biosciences) or Flow Jo Software (TreeStar, Ashland, OR, USA).
6
Isolation and Characterization of Adipose Stem Cells
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Adipose stem cells were isolated from adipose tissue collected from hospital with the consent of patient (n = 3) going through liposuction. Isolation of adipose stem cells was performed following a previous protocol with slight modifications [43 ]. Briefly, the collected sample was washed thrice with 1× PBS. Washed adipose was treated with Collagenase 1A and incubated at 37°C for 45 minutes. Digested adipose was first filtered through a 100 µm mesh, treated with active media and then centrifuged for 10 minutes at 1200×g. SVF obtained in the pallet form was resuspended in 1 ml low-glucose Dulbecco’s modified eagle’s medium (LG-DMEM) and shifted to a 75cm2 flasks. Cells from passage 2 were further seeded for experimental purposes. Moreover, passage 2 cell were also subjected to Immunophenotyping through flow cytometery for CD 90-PE, CD73-PE, CD49D-PE, CD45-FITC, CD34-PE, CD106-FITC (BD Biosciences, USA) and CD105 (Santa Cruz Biotechnology, USA) according to minimal criteria of ISCT mentioned before for defining MSCs [11 (link)]. FACS were performed according to the already published protocol [44 (link)]
7
Comprehensive Immunophenotyping of hMSCs
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Cells from passage four were used to analysis of cell surface markers. The cells were washed with PBS, and then detached from the plastic with TryPLE (Gibco Carlsbad, CA). Next, the cells were stained for CD106-FITC (clone: 51-10C9), CD73-PE (clone: AD2), CD34-PE (clone: My10), CD105-PE-CF594 (clone: 266), CD90-PE-Cy7 (clone: SE10), CD29-APC (clone: MAR04), CD14-Alexa 700 (clone: M5E2) from BD Pharmingen (San Diego – CA), CD44-PerCPCy5 (clone: G44-26), HLA-DR-APC-H7 (clone: G46-6) from Biosciences (San Jose – CA), CD45-V500 (clone: H130) and CD31-V450 (clone: WM59) from Biolegend (San Diego – CA), and for fluorescence minus one (FMO). After staining, the tubes were incubated at room temperature for 30 minutes, followed by a wash step; the cell pellet was ressuspended and measurements were performed using FACSARIA equipment (BD Biosciences).
The human mesenchymal stem cells (hMSCs) used in our assays fulfilled this identity criteria established by the International Society for Cell Therapy (ISCT), as shown byFigure S1 .
The human mesenchymal stem cells (hMSCs) used in our assays fulfilled this identity criteria established by the International Society for Cell Therapy (ISCT), as shown by
8
Immunophenotyping of Mesenchymal Stem Cells
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For immunophenotyping, MSCs were stained with fluorescein isothiocyanate- (FITC-) or phycoerythrin- (PE-) conjugated monoclonal antibodies: CD14-FITC, CD29-FITC, CD31-PE, CD34-FITC, CD44-PE, CD45-PE, CD73-PE, CD90-FITC, CD105-PE, and CD106-FITC (all from BD Pharmingen, San Diego, CA, USA). Additionally, FITC- and PE-conjugated isotype controls were used as negative controls. Briefly, cultured MSCs were harvested and stained with the antibodies for 20 min at 4°C. Subsequently, the stained cells were washed with phosphate buffered saline (PBS) and fixed with 1% paraformaldehyde (Biosesang, Seongnam, Korea). The cells were analysed using a flow cytometer (Cytomics Flow Cytometer; Beckman Coulter, Fullerton, CA, USA).
9
Phenotypic and Functional Characterization of pMSCs
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5 × 105 pMSCs per tube were phenotypically characterized by flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA), using the following antibodies CD105-PerCP, CD54-PE, CD44-FITC, CD49e-PE, CD166-PE, CD13-APC, HLA-ABC-PE, CD45-FITC, CD14-PE, CD51,61-FITC, CD106-FITC, CD34-PerCP, CD31-FITC, and HLA-DR-FITC (Pharmigen, BD Biosciences, Franklin Lakes, NJ, USA). Ten thousand events were recorded for each sample and data was analyzed using CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). In addition, pMSCs were functionally characterized by multipotent differentiation in adipocytes and osteocytes, as previously described [37 (link)].
10
Multimarker Profiling of Cell Lineages
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The primary anti-human or anti-mouse antibodies (CD29-PE, CD49a-PE, CD49b-PE, CD49c-PE, CD49d-FITC, CD49e-PE, CD49f-PE, CD51/61-PE, CD44-APC, CD45-FITC, CD47-PE, CD324-PE, CD325-PE, CD326-PE, CD54-PE, CD106-FITC, CD62E-APC, CD62L-APC, CD15s, isotype controls) and secondary antibodies were all obtained from Becton Dickinson (BD) Pharmingen™. Human interleukin-1 beta (IL-1β) was purchased from Cell Signaling Technology, Inc. 17β-estradiol (E2) and progesterone (P4) were purchased from Shanghai Yuanye biological technology Co., Ltd Mifepristone (RU-486) was purchased from Shanghai New Hualian pharmaceutical Co., Ltd with purity > 98%. Metapristone was synthesized by our laboratory with purity > 98%. Calcein-AM was obtained from Dojindo Molecular Technologies, Inc.
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