IPSC lines were rinsed with 1× PBS, fixed with 4% paraformaldehyde, and stored in PBS for staining. Cells were permeabilized with PBST (PBS supplemented with 0.1% Triton X-100) for 10 min at room temperature. Non-specific antigens were then blocked by incubating the cells with PBST+BSA at room temperature for 1 hr. Cells were incubated overnight at 4°C in PBS-BSA containing 10% donkey serum and the following specific primary antibodies: 1:500 anti-human OCT4 (Cell Signaling Technologies), 1:500 anti-human NANOG (Cell Signaling Technologies), 1:500 anti-human SSEA4 (Cell Signaling Technologies),), 1:500 anti-TRA-1–60 (Cell Signaling Technologies), 1:500 anti-SOX2 (Cell Signaling Technologies), and 1:200 anti-Lin28 (R&D Systems). Following 3 washes with PBS, cells were incubated with appropriate Alexa Fluor conjugated secondary antibodies at 1:200 dilution. After 3 washes with PBS, the samples were incubated for 10 min with DAPI (1 µg/mL) in PBS, followed by a final wash in PBS. Fluorescence images were captured at 10× or 20× on a Zeiss LSM 710/780 confocal microscope and processed using ImageJ.
Anti tra 1 60
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-TRA-1–60 is a primary antibody that specifically binds to the TRA-1-60 antigen. TRA-1-60 is a cell surface marker expressed on undifferentiated human embryonic stem cells and induced pluripotent stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti human nanog, by Cell Signaling Technology (2 mentions)
Anti sox2, by Cell Signaling Technology (2 mentions)
Anti human oct4, by Cell Signaling Technology (2 mentions)
Anti human ssea4, by Cell Signaling Technology (2 mentions)
Anti lin28, by R&D Systems (2 mentions)
Lsm 710 780 confocal microscope, by Zeiss (2 mentions)
Anti ssea4, by Cell Signaling Technology (2 mentions)
Anti nanog, by Cell Signaling Technology (2 mentions)
Slowfade diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti sox10, by Santa Cruz Biotechnology (1 mentions)
Blocking one, by Santa Cruz Biotechnology (1 mentions)
Alexa 488 or alexa 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti oct3 4, by Cell Signaling Technology (1 mentions)
Bz x700, by Keyence (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Anti sox2, by Abcam (1 mentions)
Anti tra 1 81, by Cell Signaling Technology (1 mentions)
Anti ssea1, by Cell Signaling Technology (1 mentions)
Anti h3k27me3, by Abcam (1 mentions)
5 protocols using anti tra 1 60
1
Characterization of Induced Pluripotent Stem Cells
2
Characterization of Induced Pluripotent Stem Cells
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IPSC lines were rinsed with 1× PBS, fixed with 4% paraformaldehyde, and stored in PBS for staining. Cells were permeabilized with PBST (PBS supplemented with 0.1% Triton X-100) for 10 min at room temperature. Non-specific antigens were then blocked by incubating the cells with PBST+BSA at room temperature for 1 hr. Cells were incubated overnight at 4°C in PBS-BSA containing 10% donkey serum and the following specific primary antibodies: 1:500 anti-human OCT4 (Cell Signaling Technologies), 1:500 anti-human NANOG (Cell Signaling Technologies), 1:500 anti-human SSEA4 (Cell Signaling Technologies),), 1:500 anti-TRA-1–60 (Cell Signaling Technologies), 1:500 anti-SOX2 (Cell Signaling Technologies), and 1:200 anti-Lin28 (R&D Systems). Following 3 washes with PBS, cells were incubated with appropriate Alexa Fluor conjugated secondary antibodies at 1:200 dilution. After 3 washes with PBS, the samples were incubated for 10 min with DAPI (1 µg/mL) in PBS, followed by a final wash in PBS. Fluorescence images were captured at 10× or 20× on a Zeiss LSM 710/780 confocal microscope and processed using ImageJ.
3
Immunocytochemistry of Pluripotent Stem Cells
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Cultured cells were fixed in 4% paraformaldehyde for 10 min at 4°C, permeabilized with 0.2% Triton and 50 mM NH4Cl. Fixed samples were pre-treated with BlockingOne (Nacalai Tesque) for 30 min at RT, and incubated with anti-OCT3/4, anti-NANOG, anti-SSEA4, anti-TRA1-60 (Cell Signaling Technology; diluted 1/200), anti-GFP (Molecular Probes; diluted 1/500), anti-SOX10 (SantaCruz Biotechnology; diluted 1/100) antibodies in 5% of BlockingOne in PBST for overnight at 4°C. After three washes with 0.1% Tween20 in PBS, cells were incubated with Alexa488 or Alexa594-conjugated secondary antibodies (Molecular Probes; diluted 1/500). Cells were washed and mounted in SlowFade Diamond antifade mountant with DAPI (Molecular Probes). Fluorescent images were collected on the software of BZ-X700 (Keyence). For quantitation of cultured cells, numbers of dishes were analyzed from triplicate experiments.
4
Immunocytochemical Analysis of Pluripotency Markers
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For immunocytochemical analysis, cells were fixed with 4% paraformaldehyde (PFA) in DPBS for 20 min at room temperature. Fixed cells were washed three times with DPBS, incubated in 0.2% Triton X-100 buffer for 15 min, and washed three times. After blocked in 2% BSA blocking buffer for 30 min, cells were incubated in 1% BSA buffer containing primary antibodies at 4°C overnight. The following primary antibodies were used: anti-Oct4 (Santa cruz, sc-5297), anti-Sox2 (Abcam, ab97959), anti-SSEA-1 (Cell Signaling Technology, 4744S), anti-SSEA-4 (Cell Signaling Technology, 4755P), anti-Tra-1-81 (Cell Signaling Technology, 4745P), anti-Tra-1-60 (Cell Signaling Technology, 4746P) and anti-H3K27me3 (Abcam, ab6002). The cells were washed twice in DPBS and stained for 1 h in secondary antibody, Alexa Fluor 488 (or 594) Goat Anti-Mouse IgG (H+L) or Alexa Fluor 488 (or 594) Goat Anti-rabbit IgG (H+L) (Life Technologies). For nuclear staining, the cells were incubated for 2 min with Hoechst 333342 (10 ng/ml) (Life Technologies, 1046265). The images were captured by Nikon A1 microscope.
5
iPSC Purity Validation via Flow Cytometry
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At passage 40, we validated iPSC purity based on TRA-1-60 and NANOG protein expression. We submitted the iPSCs to Accutase digestion in 6-cm plates for approximately 2.5 min at 37°C to obtain a single-cell suspension, which we washed with PBS. Next, we fixed the cells with 4% formaldehyde for 15 min at room temperature and chilled them on ice for 1 min, followed by permeabilization with 0.1% Triton X-100 for 30 min and resuspension in anti-TRA-1-60 (Cat#2372 54,746, Cell Signaling Technology, USA) and anti-NANOG (Cat#3580, Cell Signaling Technology, USA) primary antibodies. We then washed the cells with Dulbecco’s Phosphate-Buffered Saline (DPBS, Cat#21,600,010, Invitrogen, USA), followed by incubation with the corresponding species-specific fluorescence-conjugated Alexa Fluor 488-labeled goat anti-mouse IgG secondary antibody (Cat#2372A-11029, Invitrogen, USA). We performed each step at room temperature, followed by washing with DPBS. We analyzed the positively stained cells using a flow cytometer (Cat#651,155, BD FACS Verse, BD Bioscience, USA) according to the manufacturer’s protocol and performed the flow cytometry experiment and data analysis using Attune CytPix (Invitrogen, USA) and FlowJo_v10.9.0 (Flowserve, USA).
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