Immunocytochemical Analysis of Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human iPS cell-derived cardiomyocytes (iCell Cardiomyocytes^{2 (link)}) and cardiac progenitors were fixed with 4% formaldehyde in PBS and permeabilized using PBS containing 0.1% Triton X-100 (Sigma Aldrich) and 3% non-fat milk. Cardiac Troponin I (Abcam, ab56357, 1:200 dilution) and NKX2.5 (Abcam, ab35842, 1:200 dilution) were incubated in a permeabilization buffer at 4 °C overnight. The following day, nuclei were labeled with 0.1 μg/mL DAPI along with secondary antibodies (Thermo Fisher Scientific, a21467 or a11010, 1:500 dilution) in PBS. Cardiac Troponin I, NKX2.5, and DAPI were imaged accordingly on a Nikon A1R Spectral Scanning Confocal Microscope at RT.

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Zou Y., Henry W.S., Ricq E.L., Graham E.T., Phadnis V.V., Maretich P., Paradkar S., Boehnke N., Deik A.A., Reinhardt F., Eaton J.K., Ferguson B., Wang W., Fairman J., Keys H.R., Dančík V., Clish C.B., Clemons P.A., Hammond P.T., Boyer L.A., Weinberg R.A, & Schreiber S.L. (2020). Plasticity of ether lipids promotes ferroptosis susceptibility and evasion. Nature, 585(7826), 603-608.

Publication 2020

Triton X-100 ab56357 ab35842 a21467 a11010 A1R Spectral Scanning Confocal Microscope

Antibodies Buffer Cardiac Cardiomyocytes Confocal scanning microscope Dapi Dilution Formaldehyde Human ips cell Milk Nuclei Triton x 100 Troponin i

Corresponding Organization : Broad Institute

Other organizations : Whitehead Institute for Biomedical Research, IIT@MIT, Allen Institute, Harvard University

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Variable analysis

independent variables

Cell type (human iPS cell-derived cardiomyocytes and cardiac progenitors)

dependent variables

Expression of Cardiac Troponin I
Expression of NKX2.5
Nuclear staining with DAPI

control variables

Fixation with 4% formaldehyde in PBS
Permeabilization using PBS containing 0.1% Triton X-100 and 3% non-fat milk
Incubation of primary antibodies (Cardiac Troponin I and NKX2.5) at 4°C overnight
Labeling of nuclei with 0.1 μg/mL DAPI
Incubation of secondary antibodies in PBS
Imaging using a Nikon A1R Spectral Scanning Confocal Microscope at room temperature

positive controls

None specified

negative controls

None specified

Annotations

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