Cell cultures were fixed using 4% formaldehyde in phosphate-buffered saline. Primary antibodies were incubated overnight at 4 °C in labeling buffer containing 0.05 M Tris, 0.9% NaCl, 0.25% gelatin and 0.5% Triton-X-100 (pH 7.4). The following primary antibodies were used: SOX2, Nestin, MAP2, TBR1, GAD67, NeuN and glial fibrillary acidic protein (GFAP) (Merck-Millipore); FOXG1 (ProSci, Poway, CA, USA); Vimentin (Santa Cruz Biotechnology, Dallas, TX, USA); AFP (R&D Systems, Minneapolis, MN, USA); TRA-1-81 and Nanog (Beckton Dickinson, Franklin Lakes, NJ, USA); OCT4, BRN2, SATB2, CUX1, CUX2 and CTIP2 (Abcam, Cambridge, UK); Synapsin, MAP2 (Synaptic Systems, Göttingen, Germany); and PSD95 (Thermo Fisher Scientific). The following secondary antibodies were used: Alexa-488, Alexa-546, Alexa-555 and Cy3 antibodies (Jackson ImmunoResearch, West Grove, PA, USA). Samples were imbedded in Mowiol 4-88 (Sigma-Aldrich), after which confocal imaging was performed with a Zeiss LSM700 confocal microscope using ZEN software (Zeiss, Oberkochen, Germany).
Tra 1 81
Manufactured by BD
Sourced in United States
The TRA-1-81 is a laboratory instrument designed for the detection and analysis of specific cellular markers. It functions as a tool for researchers to identify and characterize cells based on the expression of the TRA-1-81 antigen. The core purpose of this equipment is to provide researchers with a reliable and precise method for studying cellular characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Tra 1 60, by BD (9 mentions)
Ssea4, by BD (7 mentions)
Ssea 3, by BD (3 mentions)
Oct3 4, by BD (2 mentions)
Antibodies for ssea4, by BD (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Ctip2, by Abcam (1 mentions)
Satb2, by Abcam (1 mentions)
Gad67, by Merck Group (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
Nestin, by Merck Group (1 mentions)
Alexa 546, by Jackson ImmunoResearch (1 mentions)
Alexa 555, by Jackson ImmunoResearch (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Psd 95, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Alexa 488, by Jackson ImmunoResearch (1 mentions)
15 protocols using tra 1 81
1
Immunofluorescence Characterization of Cell Cultures
2
iPSC Characterization and Hematopoietic Cell Sorting
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Directly conjugated monoclonal antibodies were used for iPSC characterization (Human Pluripotent Stem Cell Transcription Factor Analysis Kit, expression of stage-specific embryonic antigen [SSEA]3, eBioscience, San Diego, CA and tumor rejection antigen 1 [TRA-1]-81, Becton Dickinson, le Pont de Claix, France), and for sorting and characterization of hematopoietic cells (anti-CD34, Beckman, Villepinte, France; anti-CD43, CD42 and GPA, Invitrogen, Cergy-Pontoise, France; and anti-CD41 and CD15, Pharmingen, San Diego, CA). Cells were sorted on an Influx flow cytometer (Becton Dickinson) and analyzed on a FACS Canto II (Becton Dickinson).
3
Pluripotency Marker Expression in iPSCs
4
Barcoded Cell Profiling with FCB
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FCB was performed as previously described (Krutzik and Nolan, 2006 (link)). In brief, in a 96-well format cells were fixed for 10 min at room temperature in 4% paraformaldehyde. Following fixation, cells were resuspended in 100% methanol with fluorescent dyes (Life Technologies) and incubated at room temperature for 20 min. Cells were subsequently washed twice in PBS containing 0.1% BSA. Barcoded cells where then combined together and divided into two tubes: one tube of cells was stained with TRA-1-60 and the other was stained with TRA-1-81 (BD Biosciences, 1:1,000). Barcoded samples were then measured by flow cytometry (BD Biosciences) and all data was analyzed using FlowJo Cell Analysis software. Only cells expressing high levels of both markers and that were GFP negative were used in qPCR analysis.
5
Maintenance and Characterization of Transgenic hESCs
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hESC (AND1 cell line) were maintained undifferentiated on a layer of irradiated human mesenchymal stem cells in complete knockout Dulbecco modified Eagle medium containing 20% knockout serum replacement and 8 ng/mL basic fibroblast growth factor.34 (link),35 (link) The medium was changed daily, and cells were passaged weekly by dissociation with 1:1 collagenase IV:dispase. Cultures were visualized daily by phase contrast microscopy. Approval for hESC work was obtained from the Spanish National Embryo Ethical Committee. The pluripotency of transgenic hESC was characterized by flow cytometry using antibodies against SSEA-3, SSEA-4 TRA-1-60 and TRA-1-81 (BD Biosciences).36 (link) Expression of the pluripotency-associated transcription factors OCT4, NANOG, SOX2, CRIPTO, and DNMT3B as well as transgene expression (MA4 and A4M) were analyzed by quantitative real-time polymerase chain reaction (PCR) (Online Supplementary Table S1 shows the primers and PCR conditions used).23 (link),37 (link),38 (link)
6
Immunophenotyping of Induced Pluripotent Stem Cells
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iPSCs were selected based on morphology, subcloned for five to seven passages, and then manually picked as single-colony cells and seeded into a 96-well plate wells containing 8.5 × 103 irradiated mouse embryonic fibroblasts per well in standard hESC medium. Cells were washed with PBS, fixed at room temperature in 4% PFA/PBS for 15 min, washed with PBS, and permeabilized/blocked for 1 h at room temperature using 0.1% Triton X-100 in animal-free blocker (Vector Laboratories) followed by three PBS washes. Cells were subsequently incubated with primary antibodies (OCT4: clone 40/oct-3, Alexa Fluor 555, 1:50 dilution; NANOG: N31-355, PE, 1:30; SSEA4: MC813-70, Alexa Fluor 488, 1:200; SSEA3: MC-631, Alexa Fluor 647, 1:30; TRA-1-81: Alexa Fluor 647, 1:30; TRA-1-60: Alexa Fluor 488, 1:50; all from BD) and 2 µg/ml Hoechst 33342 in animal-free blocker at 4°C overnight, washed, and imaged on a Pathway 435 bioimager (BD) using a 10× lens. Background images (empty well images) were subtracted, and images were cropped, pseudocolored, and assembled into figure panels using the ImageJ software (National Institutes of Health). Immunofluorescence intensity was compared with a control hES cell line as internal standard to yield semiquantitative results.
7
Characterization of AMSC Phenotype
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Flow cytometry analysis was performed on AMSCs at P5. Cells (2 × 10 5 ) were incubated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) conjugated with antibodies: SSEA4, TRA-1-60, TRA-1-81, CD34, CD13, CD90, CD9, CD73, and CD105 (1:1,000; BD, Becton Dickinson, San Jose, CA, USA) for 30 min. Ten thousand events were acquired per antibody with fluorescence-aided cell sorting (FACS) in a Calibur Flow Cytometer (BD, Hia leah, FL, USA). The results were then analyzed using CellQuest Pro software (BD Biosciences, San Jose, CA, USA).
8
Pluripotency and Hematopoietic Differentiation Analysis
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K562 cells were collected, washed with cold PBS containing 0.1% Sodium azide and analyzed. hESC cells were dissociated with collagenase type IV, half of the well planted on fresh matrigel coated 12 well-plates and the rest resuspended in FACS buffer (PBS, 3% FBS, 2 mM EDTA) for analysis. The cell suspension was filtered through a 70 µm cell strainer (BD Biosciences, Bedford, MA). Live cells were identified by exclusion of the 7-AAD dye. Transduced and/or untransduced cells were stained with the antibodies SEEA-3, SSEA-4, TRA-1-60, TRA-1-81 and OCT3/4 (BD Bioscience) for pluripotency analysis. For hematopoietic differentiation, the hESCs-differentiated population were stained with anti-CD45-APC (Miltenyi Biotech, Bergish Gladbach, Germany). The different samples were analyzed using a FACSCanto II Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ) equipped with the FACSDiva analysis software (BD. Biosciences).
9
Pluripotency Validation via Flow Cytometry
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Flow cytometric analysis was used for pluripotency validation [20] . GC-ADO2-iPSCs were dissociated with Solase dissociation buffer (no.
RP01021, Nuwacell Biotechnologies Co., Ltd., Hefei, Anhui, China) and incubated with the antibodies for SSEA-4 (no. 560128, BD Pharmingen, San Diego, CA, USA) or TRA-1-81 (no. 560161, BD Pharmingen, San Diego, CA, USA) at 4 ℃ for 20 min in dark. Samples were then analyzed using a FACScan flow cytometry (BD Biosciences, San Diego, CA, USA), and data were analyzed using the FlowJo Software (version 7.6.5, FlowJo LLC, Ashland, OR, USA).
RP01021, Nuwacell Biotechnologies Co., Ltd., Hefei, Anhui, China) and incubated with the antibodies for SSEA-4 (no. 560128, BD Pharmingen, San Diego, CA, USA) or TRA-1-81 (no. 560161, BD Pharmingen, San Diego, CA, USA) at 4 ℃ for 20 min in dark. Samples were then analyzed using a FACScan flow cytometry (BD Biosciences, San Diego, CA, USA), and data were analyzed using the FlowJo Software (version 7.6.5, FlowJo LLC, Ashland, OR, USA).
10
Flow Cytometry Analysis of hPSC Markers
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Quantitative detection of hPSC-associated markers was performed using flow cytometry as previously described [16 (link),38 (link),39 (link)]. Briefly, single cells were live-stained for the cell surface markers: TRA-1-81 (BD Biosciences, #560161, San Jose, CA, USA), TRA-1-60 (BD Biosciences, #560884) and SSEA-4 (BD Biosciences, #560126, San Jose, CA, USA). Cells were also fixed, permeabilized and stained for OCT4/POU5F1 (Cell Signaling, #5177S, Danvers, MA, USA). The samples were processed using either FACSCantoTM II (Becton Dickinson) or the FACSCelestaTM (Becton Dickinson, San Jose, CA, USA), and data was acquired using the BD FACSDiva Software followed by analysis using FlowJo v10 software (FlowJo, San Jose, CA, USA).
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