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Neonatal cardiomyocyte isolation kit

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The Neonatal Cardiomyocyte Isolation Kit is a laboratory tool designed to isolate cardiomyocytes from neonatal cardiac tissue. It provides a standardized protocol and reagents to enable the extraction and purification of these cells for further analysis and research purposes.

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Lab products found in correlation

Neonatal heart dissociation kit, by Miltenyi Biotec (5 mentions) Gentlemacs dissociator, by Miltenyi Biotec (2 mentions) Lew crlcrlj, by Charles River Laboratories (1 mentions) Hank s balanced salt solution, by Fujifilm (1 mentions) Cd31 apc, by BioLegend (1 mentions) Cd45 percp, by BD (1 mentions) Gentlemacstm, by Miltenyi Biotec (1 mentions) Octo dissociator, by Miltenyi Biotec (1 mentions) Facsaria flow cytometer, by BD (1 mentions) Cd11b pe, by BD (1 mentions) Neonatal cardiac endothelial cell isolation kit, by Miltenyi Biotec (1 mentions) Red blood cell lysis solution, by Miltenyi Biotec (1 mentions) Nerve growth factor (ngf), by Thermo Fisher Scientific (1 mentions) Norepinephrine, by Merck Group (1 mentions)

9 protocols using neonatal cardiomyocyte isolation kit

1

Isolation of Embryonic and Neonatal Cardiomyocytes

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Embryonic ventricular cardiomyocytes were isolated from 14.5 days old (E14.5) hemizygous Fucci embryos and wildtype littermates (for control of background fluorescence). We used a the Neomyts cardiomyocyte isolation kit (Cellutron Life Technologies NC9073658) and neonatal cardiomyocyte isolation kit (Miltenyi Biotec 130–100-825) according to manufacturer protocols. The final pellet of cells was resuspended in 500 μL of PBS, supplemented with 5% FBS, and filtered through a 100 μm Nylon mesh to remove remaining tissue chunks. For both E14.5 and neonatal groups, single-cell analysis used samples from the same litter in each experiment, whereas the collection of pooled cells and tissue samples involved the combination of 2 litters for optimal RNA yields.
Han L., Choudhury S., Mich-Basso J.D., Ammanamanchi N., Ganapathy B., Suresh S., Khaladkar M., Singh J., Maehr R., Zuppo D.A., Kim J., Eberwine J.H., Wyman S.K., Wu Y, & Kühn B. (2020). Lamin B2 levels regulate polyploidization of cardiomyocyte nuclei and myocardial regeneration. Developmental cell, 53(1), 42-59.e11.
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2

Isolation and Culture of Neonatal Rat Cardiomyocytes

Cited 2 times
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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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3

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 gentleMACSTM 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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4

Isolation of Mouse Cardiomyocytes

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For isolation of CMs from e14-p4 timepoints, we used the neonatal cardiomyocyte isolation kit from Miltenyi Biotec in conjunction with the gentleMACS Dissociator. For later timepoints, we performed Langendorff isolation of CMs. We prepared the following buffers:
We used a horizontal (i.e. non-hanging) Langendorff apparatus with a chamber filled with perfusion buffer. To perform isolation, we first performed isofluorane anaesthesia on non-heparinized mice. Mice were observed until clearly anaesthetized and unresponsive to toe pinch, and subsequently euthanized by cervical dislocation. The heart was then rapidly excised from the chest and cannulated to the Langendorff apparatus. Flow time and rate of flow were dependent on the age of the mouse and were typically judged based on completeness of digestion to touch. Subsequently, the left ventricular free wall was excised and minced. We filtered isolated cells through a 100 μM screen to eliminate large tissue chunks, spun down at 800 RPM for 1 minute (Eppendorf centrifuge 5702), and resuspended cells in 10 mL Tyrode’s buffer.
Kannan S., Farid M., Lin B.L., Miyamoto M, & Kwon C. (2021). Transcriptomic entropy benchmarks stem cell-derived cardiomyocyte maturation against endogenous tissue at single cell level. PLoS Computational Biology, 17(9), e1009305.
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5

Isolation of Cardiac Cell Populations

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Primary cardiac cells were isolated from P1 and P7 mice using a neonatal dissociation kit (gentleMACS) according to the manufacturer’s instructions. Separation to distinct cardiac cell populations was performed by using the Neonatal Cardiac Endothelial Cell Isolation kit (130-104-183, Miltenyi Biotec), Neonatal Cardiac Fibroblast Cell Isolation kit (130-101-372, Miltenyi Biotec) or by Neonatal Cardiomyocyte Isolation kit (130-100-825, Miltenyi Biotec), according to the manufacturer’s instructions.
Bassat E., Mutlak Y.E., Genzelinakh A., Shadrin I.Y., Umansky K.B., Yifa O., Kain D., Rajchman D., Leach J., Bassat D.R., Udi Y., Sarig R., Sagi I., Martin J.F., Bursac N., Cohen S, & Tzahor E. (2017). The extracellular matrix protein agrin promotes heart regeneration in mice. Nature, 547(7662), 179-184.
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6

Isolating and Manipulating Neonatal Mouse Cardiomyocytes

Cited 1 time
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Heart tissues from WT (C57BL6/J) and MCUflox/flox mouse [16 (link)] pups at postnatal day 0 to 3 (P0–P3) were dissociated with the Neonatal Heart Dissociation Kit (Miltenyi Biotec, Auburn, CA). NMVMs were isolated from the heart cell suspension by magnetic labeling of non-cardiomyocytes with the Neonatal Cardiomyocyte Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Isolated NMVMs were cultured on fibronectin-coated coverslips, supplemented with Medium 199 containing 2% fetal bovine serum (FBS) and 100 U of penicillin G/mL, and kept in a humidified incubator at 37C with 5% CO2. When they reached 80% confluence, plated NMVMs were transfected with the mature miR-181c using Lipofectamine RNAiMAX Transfection Reagents (P/N56531, Invitrogen by Life Technologies) for 48 h. NMVMs were isolated from MCUflox/flox pups, and CRE was activated with adenovirus packaged with Recombinase Cre (Cat# 1700, Vector Biolabs) for 5 days. This adenovirus infection results in loss of MCU expression. During the last 48 h of the 5-day adenovirus treatment, we also transfected cells with either scrambled sequence or miR-181c mimic (Qiagen, Valencia, CA).
Roman B., Kaur P., Ashok D., Kohr M., Biswas R., O’Rourke B., Steenbergen C, & Das S. (2020). Nuclear-mitochondrial communication involving miR-181c plays an important role in cardiac dysfunction during obesity. Journal of molecular and cellular cardiology, 144, 87-96.
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7

Isolation of Embryonic Cardiomyocytes for ATAC-Seq

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E12.5 heart ventricles were dissociated into single cell-suspensions using the Neonatal Heart Dissociation Kit (Miltenyi Biotec #130-098-373) with minor modifications from the manufacturer’s protocol. Embryonic tissue samples were incubated twice with enzyme dissociation mixes at 37°C for 15 min with gentle agitation by tube inversions between incubations. Cell mixtures were gently filtered through a 70 μm cell strainer and centrifuged at 300 x g, 5 min, 4°C. Red blood cells were lysed with 10X Red Blood Cell Lysis Solution (Miltenyi #130-094-183) and myocytes were isolated using the Neonatal Cardiomyocyte Isolation Kit (Miltenyi Biotec #130-100-825). 75,000 isolated cardiomyocytes were used for each ATAC-Seq experiment. Libraries were prepped as previously described (Buenrostro et al., 2015 (link)).
Zhou P., Gu F., Zhang L., Akerberg B.N., Ma Q., Li K., He A., Lin Z., Stevens S.M., Zhou B, & Pu W.T. (2017). Mapping cell type-specific transcriptional enhancers using high affinity, lineage-specific Ep300 bioChIP-seq. eLife, 6, e22039.
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8

Isolation and Culture of Neonatal Mouse Ventricular Myocytes

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Heart tissue from both WT (C57BL6/J) and miR‐181c/d−/− knock‐out mice pups postnatal day 0 to day 3 (P0–P3) was dissociated using Neonatal Heart Dissociation Kit (Miltenyi Biotec, Auburn, CA). Neonatal mouse ventricular myocytes (NMVMs) were isolated from the heart cell homogenate by magnetic labeling of noncardiomyocytes using Neonatal Cardiomyocyte Isolation Kit (Miltenyi Biotec, Auburn, CA), following the company's instruction. Isolated NMVMs were cultured on fibronectin‐coated cover glasses and supplemented with Medium 199 containing 2% fetal bovine serum and 100 U of penicillin G/mL and kept in a humidified incubator at 37°C in 5% CO2.
Banavath H.N., Roman B., Mackowski N., Biswas D., Afzal J., Nomura Y., Solhjoo S., O'Rourke B., Kohr M., Murphy E., Steenbergen C, & Das S. (2019). miR‐181c Activates Mitochondrial Calcium Uptake by Regulating MICU1 in the Heart. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(24), e012919.
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9

Neonatal Cardiomyocyte Isolation and Culture

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Cardiomyocytes were isolated from P0 or P1 mouse hearts using a neonatal cardiomyocyte isolation kit (Miltenyi Biotec) based on the manufacturer’s instructions. Before plating, cardiomyocytes were filtered through a 70-μm mesh, and single cells were cultured in 24-well plates coated with gelatin. The cells were first maintained in 5% fetal bovine serum (FBS) in Dulbecco’s modified Eagle’s medium (DMEM) with penicillin/streptomycin antibiotics for 24 hours and subsequently treated for at least 2 hours with 50% horse serum in DMEM and then for 2 days with 5% FBS in DMEM. Cardiomyocytes were treated with norepinephrine (1 μΜ) (Sigma-Aldrich) or NGF (10 ng/ml) (PeproTech) as indicated.
Tampakakis E., Gangrade H., Glavaris S., Htet M., Murphy S., Lin B.L., Liu T., Saberi A., Miyamoto M., Kowalski W., Mukouyama Y.S., Lee G., Minichiello L, & Kwon C. (2021). Heart neurons use clock genes to control myocyte proliferation. Science Advances, 7(49), eabh4181.
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