Neonatal heart dissociation kit

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

The Neonatal Heart Dissociation Kit is a laboratory tool designed for the isolation and dissociation of neonatal heart tissue samples. The kit provides the necessary reagents and protocols to efficiently prepare single-cell suspensions from neonatal heart tissue for downstream applications.

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34 protocols using neonatal heart dissociation kit

Mydgf Recombinant Protein Experiments on Cardiomyocytes

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For Mydgf recombinant protein experiments, CMs were isolated from hearts of WT mice at P1 using the Neonatal Heart Dissociation Kit in combination with gentleMACS^TM and Octo Dissociator (Miltenyi BioTec, Teterow, Germany) according to the manufacturer's instructions. CMs were plated into DMEM/F12 medium supplemented with 10% FBS at 37 °C and 5% CO₂. Then CMs were treated with Mydgf recombinant protein and harvested 16 hours later, and analyzed by immunofluorescence and RNA sequencing (RNA-Seq). For inhibition of Akt, CMs were treated with Akt inhibitor LY294002 (Sigma, USA). For siRNA experiments, CMs were respectively transfected with siRNA-Akt, siRNA-c-Myc, siRNA-FoxM1 or negative control (NC) using Lipofectamine 3000 (Invitrogen, Waltham, USA) transfection reagent as previously described 22 (link), harvested 48 hours later, and analyzed by western blot, immunofluorescence, or quantitative real-time Polymerase Chain Reaction (qRT-PCR). The siRNA-Akt sequences were: 5′-GCUACUUCCUCCUCAAGAATT-3′, 5′-UUCUUGAGGAGGAAGUAGCTT-3′, the siRNA-c-Myc sequences were: 5′-CCGUACAAGCCCUAUUUCAUTT-3′, 5′-AUGAAAUAGGGCUGUACGGTT-3′, the siRNA-FoxM1 sequences were: 5′-GCAAAUUUCCAGCCGGAAUTT-3′, 5′-AUUCCGGCUGGAAAUUUGCTT-3′, and the NC sequences were: 5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′.

Wang Y., Li Y., Feng J., Liu W., Li Y., Liu J., Yin Q., Lian H., Liu L, & Nie Y. (2020). Mydgf promotes Cardiomyocyte proliferation and Neonatal Heart regeneration. Theranostics, 10(20), 9100-9112.

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Quantifying Cardiomyocyte Proliferation via SEMA6D Signaling

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Preparation of the SEMA6D conditional medium was described previously (Peng et al., 2016 (link)). Briefly, the plasmid expressing the ectodomain of human SEMA6D was transfected into 293T cells using Lipofectamine2000 (Life Technologies). The next day, the cells were washed with PBS and incubated with DMEM containing 1% FBS for 24 hours. The medium was then collected as conditioned medium. The medium isolated from cells transfected with the empty vector was included as the negative control. Cardiomyocytes from wild type E16.5 embryos were isolated using the neonatal heart dissociation kit (Miltenyi Biotec 130–098-373) following the instructions provided by the kit. Isolated cardiomyocytes were grown in DMEM (with 10% FBS, 1x glutamine, and 1x Penicillin Streptomycin) for 48 hours at 37°C in a cell culture incubator (5% CO₂). The day before EDU labeling, cells were starved overnight (1% FBS). The next day they were treated with conditioned medium harboring the ectodomain of SEMA6D or control medium. EDU was added to culture medium at a final concentration of 10uM and incubated with cardiomyocytes for 1 hour. The EDU signaling was detected using the Click-iT EdU Imaging Kit (ThermoFisher, C10337) following the manufacturer’s instructions. To distinguish cardiomyocytes from other cell types, samples were co-stained with cardiac MHC (MF20).

Sun Q., Peng Y., Zhao Q., Yan S., Liu S., Yang Q., Liu K., Rokosh D.G, & Jiao K. (2019). SEMA6D Regulates Perinatal Cardiomyocyte Proliferation and Maturation in Mice. Developmental biology, 452(1), 1-7.

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Primary Cardiac Cell Isolation and Transfection

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For primary heart cells isolation and transfection, 5–10 postnatal (PN3) hearts for each replicate (n = 4) were pooled, harvested, and kept at 37°C in culture medium (FBS 10%, 1× non-essential aminoacids, 1× PenStrep, and DMEM high glucose). Hearts were mashed with pestles for 2 min and cell isolation performed according to the manufacturer’s instructions (Neonatal Heart Dissociation Kit, Miltenyi Biotec). Cell suspension was centrifuged for 5 min at 600 × g, and cells were resuspended in cell culture medium and plated in 22.1 mm plates. 1,5 million cells were transfected 48 hr later with 75 mM si-SCR or si-Matr3 in 3 µl/ml of Lipofectamine RNAiMAX (Thermo Fisher Scientific) and 100 µl/ml of Opti-MEM (Thermo Fisher Scientific), according to the manufacturer’s specifications. Total RNA was collected 48 hr after transfection. See Supplementary file 5 for siRNA sequences. Subcellular fractionation of primary embryonal (E15.5) cardiac cells was performed using the Paris Kit (Thermo Fisher Scientific, cat#AM1921), according to the manufacturer’s instructions.

Taliani V., Buonaiuto G., Desideri F., Setti A., Santini T., Galfrè S., Schirone L., Mariani D., Frati G., Valenti V., Sciarretta S., Perlas E., Nicoletti C., Musarò A, & Ballarino M. (2023). The long noncoding RNA Charme supervises cardiomyocyte maturation by controlling cell differentiation programs in the developing heart. eLife, 12, e81360.

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Isolation and Culture of Neonatal Rat Cardiomyocytes

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Hearts were removed from LEW/CrlCrlj (Charles River Laboratories Japan, Kanagawa, Japan) or LEW-Tg (Rosa-luc)11Jmsk (Jichi Medical University, Tochigi, Japan) neonatal rats aged 0–3 days old and washed three times with Hanks' Balanced Salt Solution (HBSS; Fujifilm Wako Pure Chemical, Tokyo, Japan) [[30] (link), [31] (link), [32] (link)]. The hearts were minced into pieces less than 2 mm in size, and 7–8 pieces of tissue were placed into a gentleMACS C tube and processed in a gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Cardiomyocytes were collected using a Neonatal Heart Dissociation Kit (Milteny Biotec) and selected using a Neonatal Cardiomyocyte Isolation Kit (Milteny Biotec). The number of cardiomyocytes obtained was counted. The cells were seeded on culture dishes coated with fetal bovine serum (FBS; Thermo Fisher Scientific, Tokyo, Japan) and cultured under specific conditions for each experiment.

Yoshida A., Sekine W., Homma J., Sekine H., Itoyama Y.Y., Sasaki D., Matsuura K., Kobayashi E, & Shimizu T. (2022). Development of appropriate fatty acid formulations to raise the contractility of constructed myocardial tissues. Regenerative Therapy, 21, 413-423.

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Isolation of Cardiac Cell Types

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We isolated the mouse hearts at 1 day after AR using the Neonatal Heart Dissociation Kit in combination with gentleMACS^TM and Octo Dissociator (Miltenyi BioTec, Teterow, Germany) according to the manufacturer's instructions. Then we used the Neonatal Cardiomyocyte Isolation Kit (130100825, Miltenyi BioTec, Teterow, Germany) to isolate mouse CMs by depletion of non-target cells. Nontarget cells were directly magnetically labeled with a cocktail of monoclonal antibodies conjugated with MACS MicroBeads. The magnetically labeled non-target cells were depleted by retaining them within a MACS (MS) column in the magnetic field of a MACS Separator, while the unlabeled CMs passed through the column. As for macrophages (MΦs) and endothelial cells (ECs), we incubated the target cells for 30 minutes at 4°C with the following antibodies: CD45-Percp (1:100, BD Biosciences, 557235), CD11b-PE (1:200, BD Biosciences, 557397), CD31-APC (1:200, Biolegend, 102419). After washing, we sorted the cells by flow cytometry on a FACS Aria Flow Cytometer (BD Biosciences, San Jose, CA, USA). We identified MΦs as CD45⁺CD11b⁺, ECs as CD31⁺.

Wang Y., Li Y., Feng J., Liu W., Li Y., Liu J., Yin Q., Lian H., Liu L, & Nie Y. (2020). Mydgf promotes Cardiomyocyte proliferation and Neonatal Heart regeneration. Theranostics, 10(20), 9100-9112.

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Isolation of Neonatal Mouse Cardiomyocytes

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Single cardiomyocytes were isolated from the ventricles of mouse neonates aged from postnatal day 0 to 3 by enzymatic and mechanical dissociation in a semi-automated procedure by using the Neonatal Heart Dissociation Kit and the GentleMACS™ Dissociator (Miltenyi Biotec). Briefly, hearts were harvested, and the ventricles were separated from the atria, and digested in the GentleMACS™ Dissociator. After termination of the program, the digestion was stopped by adding medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% horse serum, 5% fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin. The cell suspension was filtered to remove undissociated tissue fragments, and centrifugated. The cell pellet was resuspended in culture medium, and the cells were plated in 60 mm-diameter Petri dishes at 37 °C for 1.5 h. The non-plated myocytes were then resuspended, plated on laminin-coated dishes at a density of 50 000 cells per plate, and incubated in 37 °C, 5% CO₂: 95% air incubator. After 24 h-plating, medium was replaced by DMEM supplemented with 1% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin, and electrophysiological experiments were performed 48 h following isolation.

Montnach J., Lorenzini M., Lesage A., Simon I., Nicolas S., Moreau E., Marionneau C., Baró I., De Waard M, & Loussouarn G. (2021). Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp. Scientific Reports, 11, 3282.

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Mouse Cardiomyocyte Isolation and miRNA Transfection

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Mice were sacrificed on d2/d3/d7 after birth; the hearts were prepared and analyzed for H2B-mCh expression by using a macroscope (AxioZoom.V16, Zeiss). Atria and ventricles of the transgenic hearts were separated and dissociated using the Neonatal heart dissociation kit (Miltenyi Biotec) without using the gentle MACS Dissociator. The heart tissue was dissected by slowly pipetting every 15 min. 7500 cells were seeded per well on a 0.001 % fibronectin-coated 384-well µ-clear microtiter plate (Greiner). Cells were incubated in differentiation medium (see above) at 37 °C and 5 % CO₂. For analysis of the binucleation index, cells were fixated after overnight adhesion to the dish.
In order to transfect postnatal CMs, ventricles of αMHC-H2B-mCh/CAG-eGFP-anillin double transgenic hearts were dissociated as described above. 5 µl of 500 nM Stock solutions of hsa-miR-199a-3p or miR mimic, Negative control #1 (Ambion, Life Technologies) were incubated with 0.2 µl Lipofectamine RNAi Max (Invitrogen) in 14.8 µl OPTI-MEM on 0.001 % fibronectin-coated 384-well µ-clear microtiter plates (Greiner) for 20 min at RT. Afterward, 7500 dissociated cells were added in a volume of 60 µl differentiation medium. Medium was changed after 48 h and the cells were further incubated for 24 h.

Raulf A., Horder H., Tarnawski L., Geisen C., Ottersbach A., Röll W., Jovinge S., Fleischmann B.K, & Hesse M. (2015). Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status. Basic Research in Cardiology, 110(3), 33.

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Isolation of Colon-26 Tumor MNC

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Tumor mononuclear cells (MNC) were isolated from Colon-26 tumor-bearing mice, 14 days after intratumoral injection with PBS or PD-H using the Neonatal Heart Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and gentleMACS Octo Dissociator (Miltenyi Biotec), according to the manufacturer’s instructions.

Hazini A., Dieringer B., Klingel K., Pryshliak M., Geisler A., Kobelt D., Daberkow O., Kurreck J., van Linthout S, & Fechner H. (2021). Application Route and Immune Status of the Host Determine Safety and Oncolytic Activity of Oncolytic Coxsackievirus B3 Variant PD-H. Viruses, 13(10), 1918.

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Isolation of Cardiac Fibroblasts from VAD Tissue

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CF were isolated from patient-derived tissue as previously published^{30 (link)}, following written informed consent under the auspices of an approved University of Rochester IRB protocol (#52958). Briefly, left ventricle tissue that would normally be discarded during ventricular assist device (VAD) implantation was freshly obtained and dissociated using a heated (37 °C) gentle-MACS Octo dissociator (Miltenyi Biotec) with an enzymatic dissociation system (Neonatal Heart Dissociation Kit; Miltenyi Biotec). The resulting cells were sedimented as described in the neonatal isolation and expanded for two passages before freezing. CFs were maintained in high-glucose DMEM supplemented with 1% penicillin–streptomycin and 10% FBS. CFs were used between passages 3 and 7 for experiments, with days to passage and passage number recorded.

Burke R.M., Lighthouse J.K., Mickelsen D.M, & Small E.M. (2019). Sacubitril/valsartan decreases cardiac fibrosis in left ventricle pressure overload by restoring PKG signaling in cardiac fibroblasts. Circulation. Heart failure, 12(4), e005565.

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Isolation and Culture of Murine Neonatal Cardiomyocytes

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Whole litters of C57BL/6JRj mice (P0) were used for isolation of mNCMs. C57BL/6JRj mice were originally obtained from Janvier labs and bred internally in CRTD animal facilities. All procedures were approved by the local ethics committee, Landesdirektion Sachsen (TVT-1/2017). P0 mNCMs were isolated using the Neonatal Heart Dissociation kit (Miltenyi Biotec, 130-098-373) following the manufacturer's instructions. Cells were seeded in plating medium (20% M199 (Thermo Fisher Scientific, 12340030), 65% DMEM (Thermo Fisher Scientific, 31966021), 5% FBS (fetal bovine serum), and 10% HS (horse serum) at a seeding density of 35,000 cells/well in a 96-well plate coated with Matrigel (Corning Life Sciences, 354234). Cells recovered for 1 day in an incubator (37°C, 5% CO₂).

Murganti F., Derks W., Baniol M., Simonova I., Trus P., Neumann K., Khattak S., Guan K, & Bergmann O. (2022). FUCCI-Based Live Imaging Platform Reveals Cell Cycle Dynamics and Identifies Pro-proliferative Compounds in Human iPSC-Derived Cardiomyocytes. Frontiers in Cardiovascular Medicine, 9, 840147.

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