Generation of the 106 iPSC lines has previously been described in detail39 (link). Briefly, cultures of primary dermal fibroblast cells were generated from a punch biopsy tissue100 (link), infected with the Cytotune Sendai virus (Life Technologies) per manufacturer’s protocol to initiate reprogramming. Emerging iPSC colonies were manually picked after Day 21 and maintained on Matrigel (BD Corning) with mTeSR1 medium (Stem Cell Technologies). Multiple independently established iPSC clones (i.e. referred to as lines) were derived from each individual. Many of the iPSC lines were evaluated by flow cytometry for expression of two pluripotent markers: Tra-1-81 (Alexa Fluor 488 anti-human, Biolegend) and SSEA-4 (PE anti-human, Biolegend)39 (link). Pluripotency was also examined using PluriTest-RNAseq39 (link). This iPSCORE resource was established as part of the Next Generation Consortium of the National Heart, Lung and Blood Institute and is available to researchers through the biorepository at WiCell Research Institute (www.wicell.org ; NHLBI Next Gen Collection). For-profit organizations can contact the corresponding author directly to discuss line availability.
Tra 1 81
Manufactured by BioLegend
Tra-1-81 is an antibody that recognizes a specific cell surface antigen. It can be used to identify and characterize certain cell types in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Tra 1 60, by BioLegend (4 mentions)
Ssea4, by BioLegend (3 mentions)
Cytoflex flow cytometer, by Beckman Coulter (2 mentions)
Alexa fluor 647 α human ssea4, by BioLegend (2 mentions)
Alexa fluor 488 ssea3, by BioLegend (2 mentions)
Cytotune sendai virus, by Thermo Fisher Scientific (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Mlc2v pe, by Miltenyi Biotec (1 mentions)
Glass bottom dishes, by Ibidi (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
Accutase, by STEMCELL (1 mentions)
Fitc labeled secondary antibody, by BioLegend (1 mentions)
Alexa fluor 488 and 568, by Thermo Fisher Scientific (1 mentions)
In cell analyzer 2000, by GE Healthcare (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti ssea 3, by BioLegend (1 mentions)
Foxg1, by Abcam (1 mentions)
6 protocols using tra 1 81
1
Generation and Characterization of iPSC Lines
2
Raman Spectroscopy of Labeled Cells
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All Raman spectra were recorded using a custom-built Raman microspectrometer as previously described (Brauchle et al., 2014 (link)). For all measurements, the laser power was maintained at 85 mW and the total accumulation time per spectrum was 100 s. Spectra were acquired from single living or fixed and antibody-labeled cells in suspension with medium or buffer in glass-bottom dishes (ibidi). For antibody-labeled cells, fluorescence microscopy was employed simultaneously with the Raman measurements to categorize cells according to their staining patterns. To discriminate single hESCs from their feeder layer, cells were stained for Tra-1-81 (mouse IgM conjugated to Alexa Fluor 488; Biolegend) without fixation (see Supplemental Information for details). For the identification of atrial and ventricular CMs, cells were fixed and labeled using MLC2a-FITC and MLC2v-PE (both Miltenyi Biotech; see Supplemental Information for details). FFPE tissues were de-paraffinized, rehydrated, and kept hydrated in medium or PBS buffer during the measurements. A background reference spectrum of the glass surface was taken every 5–10 spectra by measuring a cell-free and particle-free area.
3
Pluripotency Marker Profiling of Single Cells
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Trypsin-dissociated single cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter) and the FlowJo software program (Tree Star, CA). Expression of pluripotency markers were evaluated using the following antibodies: Alexa-Fluor®-647 α-human SSEA4 and Tra-1–81; Alexa-Fluor®-488 SSEA3 and Tra-1–60 (BioLegend). Cells were incubated for 15 min at room temperature, washed, and analyzed.
4
Immunophenotyping of Stem Cells
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Cells were dissociated using Accutase (STEMCELL Technologies) for 10 min and washed using 3% bovine serum albumin in PBS. Cells (1 × 106) were incubated with 1 μg of the indicated antibody for 1 h at room temperature. K312 was detected using a FITC-labeled secondary antibody, and phycoerythrin-labeled antibodies against SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81 (BioLegend) were used for double staining. Cell sorting was performed using a FACSAria Cell sorter (BD Bioscience).
5
Pluripotency Marker Profiling of Single Cells
6
Quantitative Immunofluorescence Assay for Cellular Reprogramming
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Cells previously fixed for 15 min at room temperature in Phosphate-Buffered Saline (PBS) with 4% paraformaldehyde were blocked with 0.1% Triton-X, 5% donkey serum, and 1% bovine serum albumin in PBS. The following primary antibodies were used in this study: SOX2 (1 : 100, Millipore), FOXG1, PAX2, HATH1 (ATOH1) (all 1 : 100, Abcam UK), PAX8 (1 : 100, Santa Cruz), POU4F3 (BRN3C, 1 : 50, Abnova), POU4F1 (BRN3A, 1 : 100, Chemicon), and B-tubulin III (1 : 100, Sigma). Secondary antibodies used were anti-mouse, anti-goat, or anti-rabbit Alexa Fluor 488 and 568 (Molecular Probes, Life Technologies, UK), while nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). Cells were imaged either on an EVOS FL Cell Imaging System or using the IN Cell Analyzer 2000 system platforms (GE Healthcare). Quantitative immunofluorescence was performed on the IN Cell Analyzer using the Developer Toolbox. Approximately 100-200 fields per antibody staining condition and per cell line were analyzed, capturing between 1,400 and 15,000 cells per condition, per line. Statistical comparison across the different antibody conditions and reprogramming methods was done using 2-way ANOVA. Results are reported as mean% ± SEM. The anti-SSEA3, SSEA4, TRA-1-60, and TRA-1-81 antibodies used for flow cytometry were from BioLegend.
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