Mouse embryos (E7.75) were dissected using forceps under a stereomicroscope (Zeiss) and regions of interest were dissociated and harvested using TrypLE. Embryoid bodies (EBs) and cells were dissociated and harvested using TrypLE. Single-cells were analyzed for RFP/GFP expression or sorted using a SH800 Cell sorter (Sony Biotechnologies). Live cells were analyzed for RFP and GFP expression and stained with antibodies targeting for the presence of appropriate markers. Cells were stained with the following antibodies: anti-mouse Cxcr4 conjugated with PerCP-eFluor 710 (1:200; 46-9991-80 eBiosciences) anti-mouse EphA2 conjugated with APC (1:100; Cat. FAB639A R&D systems), anti-human Cxcr4 conjugated with PE or APC (1:25; Cat. FAB170P R&D systems). For cTNT and Isl1 expression, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized with saponin (Sigma), stained with either mouse cTNT (1:500, Cat. MS-295-P1 Thermo Scientific) or mouse Islet1 antibody (1:200, Cat. 39.3F7 Developmental Studies Hybridoma Bank, Iowa City, IA), followed by incubation with secondary antibody conjugated with Alexa Fluor 647 (1:500, Invitrogen). Data were analyzed using FlowJo software.
Ms 295 p1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MS-295-P1 is a laboratory instrument designed for mass spectrometry analysis. It provides the core function of ionizing and detecting molecules within a sample, enabling the identification and quantification of chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Stereomicroscope, by Zeiss (1 mentions)
Percp efluor 710, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Sh800 cell sorter, by Sony (1 mentions)
Chir99021, by Merck Group (1 mentions)
α actinin, by Merck Group (1 mentions)
Guava easy cyte 8, by Merck Group (1 mentions)
Alexa fluor 647 secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ab33168, by Abcam (1 mentions)
Ab8822, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
A1r confocal microscope, by Yokogawa (1 mentions)
A12379, by Thermo Fisher Scientific (1 mentions)
Troponin t, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant containing dapi, by Thermo Fisher Scientific (1 mentions)
Elyra ps 1 confocal microscope, by Zeiss (1 mentions)
Cy 3 affinipure donkey anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Zen 2012 sp5, by Zeiss (1 mentions)
Intracellular fixation buffer, by Thermo Fisher Scientific (1 mentions)
10 protocols using ms 295 p1
1
Embryonic Stem Cell Characterization
2
Cardiomyocyte Differentiation via Temporal Wnt Modulation
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Cardiomyocyte differentiation was performed by small molecule based temporal modulation of Wnt signaling using GSK inhibitor (Gi) and Wnt inhibitor (Wi) (named as GiWi protocol) (16 (link)). Briefly, hiPSC was harvested at 80% confluence using 0.5 mM EDTA and resuspended with hiPSC-maintaining medium to 0.5×105 cells per milliliter (ml). Two ml of the cell suspension was added per well in a 12-well Matrigel-coated plate, and this is designated as day minus 4. At day 0, the cells were refreshed with RPMI/B27 medium without insulin (RPMI/B27−) containing 10 μM GSK3 inhibitor, CHIR99021 (S1263, Sigma, USA), and continued to incubate for 24 h. On day 1, the cells were refreshed with RPMI/B27− and continued to incubate for 48 h. On day 3, the cells were refreshed with RPMI/B27− containing 5 μM Wnt inhibitor, IWP2 (3533, Tocris Bioscience, UK) without insulin and continued to incubate for 48 h. On day 5, the cells were refreshed with RPMI/B27− and continued to incubate for 48 h. From day 7, the cells were refreshed with RPMI/B27 medium containing insulin (RPMI/B27＋) every 3 days till day 35. The beating cardiomyocytes can be seen as early as on day 8. The differentiated cardiomyocytes were then characterized by immunofluore-scence for the expression of cTNT (MS-295-P1, Thermo Fisher Scientific, USA) and α-actinin (A7811, Sigma, USA), and by flow cytometry for cTNT expression.
3
Flow Cytometric Analysis of Cardiomyocytes
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Differentiated cells were dissociated into single cells with 0.25% trypsin-EDTA and fixed in 1% paraformaldehyde for 10 minutes. The anti-cardiac troponin T (TNNT2) antibody (MS-295-P1, Thermo Fisher, USA) and Alexa Fluor 647 secondary antibody (715-605-151, Jackson ImmunoResearch, USA) were used for flow cytometry analysis. Data were acquired and analyzed using Guava easyCyteTM 8 (EMD Millipore, Germany).
4
Cardiomyocyte Immunostaining and Morphometrics
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Isolated cells were fixed by 4% PFA at room temperature (r.t.) for 15 min. Fixed cells were pelleted, washed two times in 1 ml of PBS, then re-suspended in permeabilization buffer at r.t. for another 15 min. Permeabilized cells were pelleted, washed two times in 1 ml of PBS, then processed for blocking and immunostaining for cardiomyocyte marker (cTnT, 1:200; Thermo Fisher Scientific, MS-295-P1). DAPI was used to counterstain nuclei. Images were acquired for analysis within 48 h. Cell length and width were determined from phase contrast images using the ImageJ plugin Coli-Inspector. Length and width measurements from ≥ 300 cells from ≥ 3 biological replicates were used to generate data for each condition.
5
Immunostaining of Vascular and Cardiac Cells
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Spiral and straight vessels, after culture for the designated time, were fixed by treatment with 4% formaldehyde for 15 min (for PDMS devices) or 60 min (for collagen devices). Devices were permeabilized by incubation in phosphate-buffered saline (PBS) containing Triton X-100 (0.5%) and bovine serum albumin (BSA; 2%) for 30 min. Unconjugated primary antibodies were added into the spirals (and onto collagen gels), incubated overnight at 4°C, followed by washing with PBS for three times (5 min for each time), and incubated with secondary antibodies and Hoechst 33342 (40 μg/ml) for 1 hour at room temperature before a final round of washing. Devices were stained for factors including CD31 (1:30; Abcam ab28364), VE-cadherin (1:100; Abcam ab33168), von Willebrand factor (1:100; Abcam ab8822), cTnT (cardiac troponin T) (1:100; Thermo Fisher Scientific MS-295-P1), and F-actin (1:100; Thermo Fisher Scientific A12379). Stained devices were imaged using a Nikon A1R confocal microscope or a Yokogawa W1 spinning disc confocal system.
6
Immunostaining of Neonatal Cardiac Myocytes
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Neonatal cardiac myocytes in 4-well chambers were washed with phosphate-buffered saline (PBS) three times, fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.3% Triton X-100 for 10 min and blocked with 10% normal goat serum for 1 h at room temperature. The following antibodies were used as primary antibodies: KLF15 (1: 200, ab185958, abcam) and Troponin T (1: 400, MS295P1, Thermo Scientific). Alexa Fluor 488 Dye-conjugated or Alexa Fluor 555 Dye-conjugated secondary antibody (Invitrogen) was used for detecting indirect fluorescence. Slides were mounted with Prolong Diamond Antifade Mountant containing DAPI (Life Technology).
7
Immunofluorescence Staining for Lamin A/C and Troponin T
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The cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Then they were washed three times with PBS 1x for 5 min. Next, the cells were permeabilized with 0.3% Triton diluted in 1x PBS for 15 min. Subsequently, blocking was done using a 5% BSA solution diluted in PBS 1x for 60 min. We then placed the primary antibody in question in PBS-BSA 3% at 1:100 (LMNA, cat. MA3-1,000, ThermoFisher scientific) for Lamin A/C; and 1:200 (Troponin T, cat. MS-295-P1, ThermoFisher scientific) for Troponin T, cardiac isoform, dilutions, and incubated overnight at 4°C. The following day, we washed 3x with 1x PBS for 5 min. After the washes, we placed the secondary antibody Cy™3 AffiniPure Donkey Anti-Mouse IgG (H + L) (cat. 715150, Jackson ImmunoResearch) diluted in PBS-BSA 3% at a 1:400 ratio for 2 h at room temperature. After incubation, we washed 3x with 1x PBS for 5 min. Finally, we placed DAPI (cat. D9542, Sigma-Aldrich) for 5 min and washed 3x times with PBS 1x for 5 min. The coverslips were sealed using 13 µL of Fluoroumont (ThermoFisher scientific). Images were taken with the ×100 objective on the Elyra PS.1 confocal microscope (Carl Zeiss), ZEN 2012 SP5 software (Carl Zeiss), at the Advanced Microscopy Unit (UMA) of the National Center for Structural Biology and Bioimaging (UFRJ, Rio de Janeiro).
8
Cardiomyocyte Isolation from Adult Hearts
9
Cardiac Troponin T Expression Analysis
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On the 14th day, differentiated cells were dissociated with 0.25% trypsin–EDTA. For intracellular staining, cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and permeabilized with 0.3% Triton X-100 in PBS for 30 minutes. Cells were blocked with 0.5% (v/v) BSA in PBS and stained with troponin T cardiac isoform Ab-1 (MS-295P1, diluted 1:200; Thermo Scientific™) for 30 minutes at room temperature. Subsequently, cells were incubated for 30 minutes at room temperature with Alexa Fluor® 647-conjugated goat anti-mouse (A21236, diluted 1:1000; Life Technologies). Permeabilized cells were selected by DAPI staining and samples were analyzed using BD FACSAria IIu and FlowJo v10 software.
10
Immunofluorescence Characterization of hiPSC-CMs
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Well-suspended hiPSC cells and induced cardiomyocytes were seeded in a μ-Slide 8 well (80827, ibidi, USA) pre-coated with Matrigel (1：100) at the density of 2×104 per well. 48 h later cells were fixed with 4% (w/v) Paraformaldehyde (PFA) for 15 min at room temperature, permeabilized, and incubated with following first primary antibodies: anti-OCT4 antibody (#2750, Cell Signaling Technology, USA), anti-NANOG antibody (#3580, Cell Signaling Technology, USA), anti-SSEA4 antibody (#4755, Cell Signaling Technology, USA), anti-TRA-1-60 antibody (#4746, Cell Signaling Technology, USA), anti-MKI67 antibody (ab15580, abcam, USA), anti-NKX2-5 antibody (ab91196, abcam, USA), anti-cTNT antibody (MS-295-P1, Thermo Fisher Scientific, USA) and anti-α-ACTININ antibody (A7811, Sigma, USA), followed by their corresponding fluorescence-labeled secondary antibodies, alexa fluor 488 labeled goat anti-rabbit IgG (A-11008, Invitrogen, USA), alexa fluor 488 labeled goat anti-mouse IgG (A-11001, Invitrogen, USA), alexa fluor 594 labeled goat anti-rabbit IgG (R37177, Invitrogen, USA) and alexa fluor 594 labeled goat anti-mouse IgG (A-11005, Invitrogen, USA). The slides were then counterstained using 0.5 μg/ml of DAPI (#4083, Cell Signaling Technology, USA) for 15 min at room temperature. After rinsing with PBS, the chambers were mounted and visualized by fluorescence microscopy (IX83, Olympus, Japan).
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