NRCs were transiently transfected with GFP-tagged C3C6 constructs using Escort III (Sigma) as described previously (31 (link)) and then fixed and permeabilized as aforementioned. Immunostaining was carried out using primary antibodies against mouse antisarcomeric alpha-actinin (Sigma) and rabbit anti MyBP-C (a generous gift from Prof Mathias Gautel, Kings College London) and secondary antibody/counterstain solution (Cy3-goat anti-mouse, catalog no.: 115-165-146; Jackson Immunochemicals) and Atto 647N-goat anti-rabbit (Sigma). Cells were mounted using 70% glycerol/PBS according to the antibody manufacturer instructions. Samples were imaged using a STEDYCON (Abberior) attached to a Leica TCS SP5 confocal microscope equipped with 594 nm and 640 nm excitation lasers and a 775 nm depletion laser using a 100×/numerical aperture 1.4 oil immersion lens. Images were recorded at a pixel size of 20 nm.
Escort 3
Manufactured by Merck Group
Sourced in United States, China
The Escort III is a lab equipment product by Merck Group. It is designed to provide accurate and reliable measurements in a laboratory setting. The core function of the Escort III is to perform precise measurements and data collection.
Automatically generated - may contain errors
Lab products found in correlation
Gelatin, by Merck Group (2 mentions)
Mouse anti sarcomeric alpha actinin, by Merck Group (1 mentions)
Stedycon, by Abberior (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Escort 4, by Merck Group (1 mentions)
Pegfp c2, by Takara Bio (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Axio scope a1, by Zeiss (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Neonatal cardiomyocyte isolation system, by Worthington (1 mentions)
7 protocols using escort 3
1
Fluorescent Imaging of Sarcomeric Proteins
2
Immunostaining and Microscopy of Cell Lines
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NRCs were prepared according to [31 (link)], and C2C12 and HEK293 cells were prepared following standard protocols. NRCs, C2C12 and HEK293 cells were transfected using Escort III (Sigma), Lipofectamine 3000 (Invitrogen) and Escort IV (Sigma), respectively, according to manufacturer’s instructions. Following incubation at 37 °C, 5% CO2 for 24–48 h for NRCs and HEK293 cells and up to 14 days for C2C12 myoblasts to allow differentiation into myotubes, cells were prepared according to [12 (link)]. Cells were stained with primary antibodies against myosin heavy chain (clone A4.1025) [9 (link)], obscurin domain O59 [61 (link)], titin Z1Z2 [15 (link)], p62/SQSTM1 (Abcam, ab41116161) and ubiquitin (Merck, FK2, 04–263) followed by Cy3 or Cy5-conjugated goat anti-rabbit or anti-mouse IgG (H + L) secondary antibodies (Jackson ImmunoResearch ML 115–165-146, 115–175-146, 111–175-144 and BioRad STAR36D549GA) and imaged on an LSM510 (Zeiss) or SP5 (Leica) confocal microscope. Antibodies were diluted 1:100 prior to incubation.
3
Neonatal Rat Cardiomyocyte Isolation and Transfection
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NRC isolation, culture, transfection, and staining were performed essentially as described previously (Pernigo et al., 2010 (link)). Briefly, NRCs were transfected with GFP-tagged transiently expressing constructs (pEGFPC2-, Clontech Laboratories) using Escort III (Sigma-Aldrich). After 48 hr culture to promote protein expression, cells were fixed with 4% paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100/PBS, and then stained with the appropriate antibodies. The antibodies used for the current work were as follows: MyB4, a mouse monoclonal antibody to the myomesin domain My12 (Grove et al., 1984 (link)); and Ob5859, a rabbit polyclonal antibody to two consecutive Ig domains in obscurin, Ob58 and Ob59 (Fukuzawa et al., 2005 (link), Young et al., 2001 (link)). All fluorescent-conjugated secondary antibodies were purchased from Jackson ImmunoResearch. All images for ratiometry analysis were collected on a Zeiss LSM510 confocal microscope as described previously (Fukuzawa et al., 2008 (link)). Image analysis was carried out as described in our previous work (Pernigo et al., 2015 (link)). Further details are available in the Supplemental Experimental Procedures .
4
Transfection Efficiency Evaluation
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Vero cells were transfected using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA) or VeroFect (OZ Biosciences, San Diego, CA, USA), or EscortIII (Sigma, St. Louis, MO, USA) according to the manufacturers’ recommendations. Cells were incubated in a 96-well plate at a concentration of 2 × 105 cell/mL in DMEM growth medium without antibiotics. After 24 h, a mixture of test plasmids and transfecting agents was added to the cells in a 1:2 ratio. The efficiency of transfection was assessed by GFP fluorescence after 48 h using an AxioScopeA1 inverted fluorescent microscope (Zeiss, Oberkochen, Germany). The ratio of the number of fluorescent cells expressing GFP to the total number of cells in the population was calculated and expressed as a percentage.
5
Isolation and Culture of Neonatal Rat Cardiomyocytes
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Primary cultures of NRCs were isolated from 1- or 2-day-old neonatal Sprague–Dawley rats. Hearts were collected and atria were removed. Hearts were then washed, excised, minced and enzymatically digested at 37 °C with ADS buffer [116 mmol/L NaCl, 20 mmol/L HEPES, 0.8 mmol/L NaH2PO4, 5.6 mmol/L glucose, 5.4 mmol/L KCl, 0.8 mmol/L MgSO4] containing collagenase (57.5 U/mL) and pancreatin (1.5 mg/mL). The suspension was pre-plated to remove contaminating cells, before being cultured on gelatin (Sigma) pre-coated 35 mm petri dishes with a density of 2 × 105 cells/ml. Cells were allowed to adhere for 24 h, and then transfected using Escort III (Sigma) following the manufacturer’s instruction as described previously (69 (link)).
6
Neonatal Rat Cardiomyocyte Isolation Protocol
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Neonatal rat cardiomyocyte (NRC) isolation was performed using a Worthington Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp) following the manufacturer's instructions. NRCs were isolated from 1- or 2-day-old neonatal Sprague–Dawley rats. Hearts were then washed, excised, minced and enzymatically digested at 37 °C with ADS buffer [116 mmol/L NaCl, 20 mmol/L HEPES, 0.8 mmol/L NaH2PO4, 5.6 mmol/L glucose, 5.4 mmol/L KCl, 0.8 mmol/L MgSO4] containing collagenase (57.5 U/ml) and pancreatin (1.5 mg/ml). The suspension was pre-plated to remove contaminating cells, before being cultured on gelatin (Sigma) pre-coated 35 mm petri dishes with a density of 2 × 105 cells/ml. Cells were allowed to adhere for 24 h, and then transfected using Escort III (Sigma) following the manufacturer’s instruction as described previously (29 (link)).
7
siRNA Knockdown and Adenoviral Infection in ECs
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For siRNA knockdown, ECs were transfected with human MIG6 siRNA (5′-CUACACUUUCUGAUUUCAA-3′) (Liu et al., 2012 (link)), human SHC1 siRNA (5′ CUACUUGGUUCGGUACAUGGG-3′) (Lundgren et al., 2006 (link)), or non-targeting scrambled negative control (Ribobio, Guangzhou, China) using ESCORT III (L3037, Sigma). For adenoviral infection, ECs were infected with Ad-MIG6 or Ad-GFP at an MOI of 10 for 48 h.
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