Analysis of the cell surface antigen scenarios was done by using the cells obtained from the third passage. The isolated cells were incubated for 10-30 minutes in a dark environment with the following anti-human antibodies: CD90- fluorescein isothiocianate (FITC), CD 34- phycoeritrin (PE), CD 45- Peridinin chlorophyll protein (PerCP), CD 271-FITC (MiltenyiBiotec), CD106-PE, CD 44-FITC, CD73-PE, CD14-FITC (BD Biosciences), and CD105-PerCP (AbDSerotec, Kidlington, Oxford, England). Isotype-matched irrelevant monoclonal antibodies such as Mouse IgG1-PE, IgG2a-FITC (AbDSerotec, Kidlington, Oxford, England), IgG2a-PErCP, IgG2b-FITC and IgG2a-PE (Miltenyi Biotec) were used to omit the effect of non-specific cells staining. Flow cytometry analysis was done in a FACS Calibur device (BD Biosciences) by using the cell quest as data acquisition software. Win MDI software, version 8.2, was employed for data analysis.
Cd271 fitc
Manufactured by Miltenyi Biotec
Sourced in Germany
CD271-FITC is a fluorescently-labeled antibody that targets the CD271 surface antigen. It is used for the identification and enumeration of cells expressing CD271 in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd106 pe, by BD (1 mentions)
Facscalibur device, by BD (1 mentions)
Cd14 fitc, by BD (1 mentions)
Igg2a pe, by Miltenyi Biotec (1 mentions)
Igg2a fitc, by Bio-Rad (1 mentions)
Cd 45 peridinin chlorophyll protein percp, by Miltenyi Biotec (1 mentions)
Igg2a percp, by Miltenyi Biotec (1 mentions)
Cd73 pe, by BD (1 mentions)
Cd44 fitc, by BD (1 mentions)
Macs separation kit, by Miltenyi Biotec (1 mentions)
Cd34 pe, by BD (1 mentions)
Dmem f12 culture medium, by Thermo Fisher Scientific (1 mentions)
Cd14 fitc, by Thermo Fisher Scientific (1 mentions)
Cd31 fitc, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Cd45 fitc, by BD (1 mentions)
Cd166 pe, by BD (1 mentions)
Cd133 apc, by Miltenyi Biotec (1 mentions)
4 protocols using cd271 fitc
1
Cell Surface Antigen Characterization
2
Isolation and Characterization of CD271+ Bone Marrow Cells
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Density gradient centrifugation was performed to obtain bone marrow mononuclear cells (BMMNCs). CD271+ cells were selected using MACS separation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD271+-selected cells were cultured in 6-well plates at low density for 10 days and analyzed by flow cytometry for the expression of CD271 and alkaline phosphatase (ALP, TRA-2-49). The monoclonal antibodies are CD271-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) and TRA-2-49-PE (Biolegend, San Diego, CA, USA).
3
Synovial Mesenchymal Stem Cell Isolation
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The collected synovial tissue was washed with 1× PBS supplemented with 10% penicillin/streptomycin (PS), followed by mechanical disintegration with a scalpel in 60 × 15 mm Petri dishes. Seeding by explant was performed, in which the SM fragments were added to DMEM-F12 culture medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 20% fetal bovine serum (FBS) and 1% PS. The explants were incubated at 37 °C in an atmosphere of 95% humidity and 5% CO2. At 24 h, nonadherent cells were removed by replacing the medium with fresh culture medium; this process was conducted every third day until the cells were 80–90% confluent. Subcultures were performed, and in the second passage, the cells were immunophenotyped using flow cytometry (BD FACSCalibur) with anti-CD90 antibodies coupled to fluorescein isothiocyanate (FITC; BD Pharmingen 561988, San Diego, CA, USA), CD105 coupled to phycoerythrin (PE; BD Pharmingen 560839), CD73 coupled to peridinin chlorophyll (PerCP-C; BD Pharmingen 581280), CD117 allophycocyanin (APC; BD Pharmingen 341106), CD14–FITC (Thermo Scientific 1-82074, Waltham, MA, USA), CD34–PE (BD Pharmingen 555822), CD45–FITC, (BD Pharmingen 555482), CD166–PE (BD Pharmingen 559263), CD271–FITC (Miltenyi Biotec 130098, Bergisch Gladbach, Germany), and CD31–FITC (Thermo Scientific 1-80360).
4
Multicolor Flow Cytometry for Protein Expression
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BD LSR II, multicolour flow cytometry was used to measure protein expression, as previously described [32] , although with the following modifications. Automatic software compensation was performed to minimise spectral overlap between different fluorochromes, and CST beads were run prior to each sample measurement to control for consistency in machine perfor-mance. The following antibodies were used: ALDH1A1-PE (Lot: HG09MY1304, Clone: 03) (Sino Biological Inc., North Wales, PA, USA), Nestin-PE (Lot: 2524561, Clone: 10C2) (Merck Millipore, Temecula, CA, USA), ABCG2-PE (Lot: B143287, Clone: 5D3) (Biolegend, San Diego, CA, USA), CD44v7/8-FITC (Lot: 150715, Clone: VFF-17) (Acris Antibodies, San Diego, CA, USA), CD44v10-FITC (Lot: 9E08V1) (Bioss, Woburn, MA, USA), CD133-APC (Lot: 5150611303, Clone: AC133) and CD271-FITC (Lot: 5150609183, Clone: ME20.4-1.H4) (both from Miltenyi Biotec, Teterow, Germany). The DNA-binding dye ethidium monoazide bromide (Biotium, Hayward, CA, USA) was used to exclude dead cells (incubated on ice under bright light for 20 min) before antibody staining. Data were analysed using FlowJo software version 10.0.7 (Tree Star, Ashland, OR, USA). Cell viability was determined by a commercial viability kit, according to the manufacturer's instructions (BD Biosciences, Heidelberg, Germany).
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