The cells were dissociated into single-cell suspensions by incubation with trypsin-EDTA solution (Thermo Fisher Scientific; Waltham, MA, US), at 37°C for 5 min, followed by centrifugation at 350 g for 5 min at room temperature. The cells were counted and stained with an APC-conjugated anti-TRA1-60 antibody (BD Biosciences; Franklin Lakes, USA) for pluripotency evaluation. The efficiency of cardiac differentiation was evaluated by staining with anticardiac troponin T (Thermo Fisher Scientific; Waltham, MA, US), after permeabilization with 0.3% Triton X-100 solution (Sigma-Aldrich; St. Louis, MI, United States). The secondary antibody anti-mouse IgG conjugated with Alexa Fluor 647 was then used (Thermo Fisher Scientific; Waltham, MA, US). Data acquisition was performed with the LSR Fortessa flow cytometer (BD Biosciences; Franklin Lakes, USA) using the FACSDiva v.6.3 acquisition and analysis software.
Anti cardiac troponin t
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-cardiac troponin T is a laboratory reagent used for the detection and quantification of cardiac troponin T in biological samples. Cardiac troponin T is a protein found in the heart muscle and its presence in the blood can be an indicator of myocardial injury.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Triton x 100 solution, by Merck Group (1 mentions)
Fluoview 1000, by Olympus (1 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Anti α actinin, by Merck Group (1 mentions)
Blocking buffer, by Solarbio (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti ph3, by Thermo Fisher Scientific (1 mentions)
Anti ki67, by Cell Signaling Technology (1 mentions)
Anti aurora b, by Abcam (1 mentions)
Anti flag, by Merck Group (1 mentions)
Spinning disk microscope, by Zeiss (1 mentions)
Anti tubb3, by BioLegend (1 mentions)
Ab97959, by Abcam (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti pdgfrα, by Cell Signaling Technology (1 mentions)
Anti yap, by Cell Signaling Technology (1 mentions)
Anti gfp, by Cell Signaling Technology (1 mentions)
8 protocols using anti cardiac troponin t
1
Pluripotency and Cardiac Differentiation
2
Immunostaining of Cardiac Cells
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The NRVMs were fixed with 4% paraformaldehyde for 10 min. The fixed cells and tissue sections were treated with Triton (0.1%) for 8 min and blocked with blocking buffer (Solarbio) for 30 min at 37°C. Then, the samples were incubated with the primary antibodies overnight at 4°C. After washing by PBS, the samples were incubated with fluorescent secondary antibodies for 1 h at 37°C, followed by 5 min of DAPI staining. After staining, microscopy was performed using Olympus confocal microscope (FluoView 1000). The primary antibodies used were anti-cardiac troponin T (#MA5-12960, Thermo Fisher Scientific), anti-α actinin (#A7811, Sigma), anti-Ki67 (#9027S, Cell-Signaling Technology), anti-pH3 (#PA5-17869, Thermo Fisher Scientific), and anti-Aurora B (#ab2254, Abcam). The secondary antibodies used were goat anti-mouse IgG (H+L), Alexa Fluor Plus 488 (#A-32723, Thermo Fisher Scientific), Goat anti-rabbit IgG (H+L), and Alexa Fluor Plus 488 (#A-11035, Thermo Fisher).
3
Immunostaining of Pluripotency and Lineage Markers
4
Protein Extraction and Western Blot
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Tissue or cells were homogenized in lysis buffer containing 50 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 1% IGEPAL CA-630, 0.1% SDS, 0.5% deoxycholic acid, 1 mmol/L EDTA, 0.1 mmol/L Na3VO4, 1 mmol/L NaF, 50 μmol/L phenylmethylsulfonyl fluoride (PMSF), 5 μg/mL aprotinin, and 5 μg/mL leupeptin. Following SDS-PAGE, immunoblotting was performed using the following antibodies: anti-YAP (Cell Signaling #14074, 1:1000), anti-cardiac troponin T (Thermo Fisher MA5-12960, 1:2000), anti-GFP (Cell Signaling #2956, 1:1000), anti-GAPDH (Cell Signaling #5174, 1:1000), anti-PDGFRα (Cell Signaling #3174, 1:1000), anti-Hsp47 (Enzo Life Sciences #ADI-SPA-470, 1:1000), and anti-tubulin (Sigma, T-6199, 1:1000). Densitometry was performed using NIH ImageJ software.
5
Isolation and Characterization of Cardiac Cells
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Wild-type Sprague–Dawley rats were purchased from Japan SLC (Shizuoka, Japan).
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Women's Medical University. The rats were anesthetized using 3%–5% sevoflurane, in compliance with ARRIVE guidelines [19 ].
The following antibodies were used for immunocytochemistry and magnetic-activated cell sorting (MACS): anti-rat CD31 (clone TLD-3A12; Bio-Rad, Hercules, CA), anti-cardiac Troponin T (#MA5-12960, Thermo Fisher Scientific, Waltham, MA), and anti-Vimentin mouse monoclonal antibodies (#Ab-8069, Abcam, Cambridge, UK), and anti-CD90 (#Ab-226, Abcam), Hoechst33258 (#H3569, Thermo Fisher Scientific) and anti-Lypd-1 (#Ab-157516, Abcam) rabbit polyclonal antibodies. Alexa Fluor 488 secondary antibody anti-mouse IgG was purchased from Thermo Fisher Scientific and all other secondary antibodies were purchased from Jackson Immuno-Research Laboratories (West Grove, PA). The MACS system including a magnet column and magnet beads were purchased from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany). Collagenase type 2 was purchased from Worthington Biochemical (Lakewood, NJ).
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Women's Medical University. The rats were anesthetized using 3%–5% sevoflurane, in compliance with ARRIVE guidelines [19 ].
The following antibodies were used for immunocytochemistry and magnetic-activated cell sorting (MACS): anti-rat CD31 (clone TLD-3A12; Bio-Rad, Hercules, CA), anti-cardiac Troponin T (#MA5-12960, Thermo Fisher Scientific, Waltham, MA), and anti-Vimentin mouse monoclonal antibodies (#Ab-8069, Abcam, Cambridge, UK), and anti-CD90 (#Ab-226, Abcam), Hoechst33258 (#H3569, Thermo Fisher Scientific) and anti-Lypd-1 (#Ab-157516, Abcam) rabbit polyclonal antibodies. Alexa Fluor 488 secondary antibody anti-mouse IgG was purchased from Thermo Fisher Scientific and all other secondary antibodies were purchased from Jackson Immuno-Research Laboratories (West Grove, PA). The MACS system including a magnet column and magnet beads were purchased from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany). Collagenase type 2 was purchased from Worthington Biochemical (Lakewood, NJ).
6
Cardiac Differentiation Protocol
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Single-cell suspensions were obtained by dissociating EBs with 0.025% trypsin for 15 min at 37°C. The cells were then fixed with 4% paraformaldehyde for 15 min and washed twice with phosphate buffered saline (PBS). The fixed cells were first permeabilized in permeabilization buffer (0.2% Triton X-100 in PBS) for 30 min and then blocked with 10% goat serum for 25 min. Cells were then incubated with the primary antibody (anti-cardiac troponin T; Thermo Fisher Scientific). After 1 hr, the cells were washed in PBS, incubated with Goat anti-mouse IgG1 - Alexa 488 secondary antibody for 45 min, and finally washed twice with PBS. All procedures were performed at 4°C. Fluorescence-activated cell sorting analysis was carried out using a BD LSR analyzer (BD Biosciences).
7
Comprehensive Immunohistochemical Analysis of Heart Tissue
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Immunohistochemistry was performed as described previously31 (link)–33 (link). Briefly, heart tissue sections were permeabilized with PBS containing 0.5% Triton X-100 for 15 min at room temperature. Tissue sections were then blocked with 1% BSA in PBS for 60 min at room temperature and incubated with primary antibodies at 4 ℃ overnight. On the next day, samples were washed three times with PBS and incubated with secondary antibodies for 1h at room temperature. DAPI solution (VectaShield) was added for nuclear staining. The primary antibodies used in this study included mouse anti-Vimentin (Millipore; 1:100), rabbit anti-α smooth muscle actin (Abcam; 1:100), anti-cardiac Troponin T (Thermo; 1:100), rabbit anti-CD31 (Abcam; 1:100), mouse anti-CD68 (Abcam; 1:200), rabbit anti-CD206 (Abcam; 1:200), mouse anti-α sarcomeric actinin (Sigma; 1:100), and mouse anti-GFP (Thermo; 1:100). Secondary antibodies used in this study included either anti-mouse/-rabbit IgG Alexa Fluor 488 (Invitrogen; 1:500) or anti-mouse/-rabbit IgG Alexa Fluor 568 (Invitrogen; 1:500). Imaging of heart sections was performed using a Laser Scanning Microscope LSM 880 NLO with Airyscan (Zeiss).
8
Immunohistochemical Analysis of Human Ventricular Tissue
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Human ventricular samples were processed as previously described.23 , 24 Briefly, they were fixed in 4% PFA (Santa‐Cruz) in PBS (Lonza) and processed for paraffin embedding. 6 μm thick sections were de‐waxed and rehydrated. Antigen retrieval was performed with Dako target retrieval solution citrate pH6 at 90°C. Human sections were incubated with anti‐GCN5 (1:100; Cell Signaling, #3305, rabbit), anti‐4HNE (1:200; Abcam, ab46545, rabbit) and anti‐cardiac Troponin T (1:300; ThermoFisher Scientific, #MA5‐12960, mouse) at 4°C overnight. After washing, sections were incubated with the appropriate fluorochrome‐conjugated secondary antibody Alexa 488 (A11034, 1:200) or Alexa 633 (A221126, 1:200) (AlexaFluor) for 1h at room temperature in the dark. Nuclear staining was performed by incubating sections with Hoechst 33342 (1:1000, ThermoFisher Scientific). Images were acquired with a confocal microscope (Zeiss LSM710—ConfoCor3 LSM, Zeiss) using the software Zen 2008 (Zeiss) and quantified with the software AxioVision Rel. 4.8.
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