H9c2 cell culture protocol pdf

To simulate ischemia, the cell culture medium for hypoxia was deprived of serum. Cell culture the rat myocardiumderived cell line h9c2 was obtained from the american type culture collection atcc, rockville, md, usa and maintained in dmem containing 4500 mgl glucose, 10% fbs, 10 mm hepes sigmaaldrich, st louis, mo, usa and a 1% penicillinstreptomycin solution at 37c in a 5% co 2 incubator thermo, waltham, ma, usa. Doxorubicin is key for the cardiotoxicity of fac 5. Manassas, va, were cultured in dmem invitrogen gibco, carlsbad, ca supplemented with 10%. The embryonic cardiomyocyte cell line h9c2 is commonly used in. Metformin protects h9c2 cardiomyocytes from highglucose and. I have started to culture my h9c2 cells in the following media. In a microbiological safety cabinet, hold a tissue soaked in 70% alcohol around the cap of the frozen ampoule and turn the cap a quarter turn to release any residual liquid nitrogen that may be trapped. H9c2 transfection reagent embryonic myocardium altogen. I have a problem with my h9c2 cell culture, i hope somebody could help me with this,i would be most grateful. I am working with h9c2 cell line and i see difference in morphology of this cells during the culturing. Perform a viable cell count when you resuscitate and harvest cell cultures. Alpinate oxyphyllae fructus alpinia oxyphylla miq extracts. The h9c2 line of embryonic rat cardiomyocytes is a subclonal line of the original clonal cell line derived from embryonic bd1x rat heart tissue by kimes and brandt.

Curcumin protects h9c2 cardiomyocyte against ischemia. For transient transfection of h9c2 cells, h9c2 cardiomyoblasts were seeded onto a sixwell plate and cultured in dmem supplemented with 10% fbs. Quantitative proteomics and systems analysis of cultured h9c2. H9c2 cells from rat embryonic ventricular myocardium and neonatal rat cultured cardiomyocytes were used in the study. Cell culture basics handbook thermo fisher scientific. Animal cell culture protocol aseptic technique and good cell culture practice to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Cell culture and treatments rat h9c2 cardiomyoblast cells were cultured in dulbeccos modified eagles medium dmem supplemented with 10% fetal bovine serum fbs, 100 iuml penicillin, 0. Im using dmem high glucose medium 4nm llactate and im having the. Cell culture h9c2 cardiac muscle cell line was obtained from american type culture collection manassas, va, usa at passage 14 and cultured in dmem complete culture medium dmemf12 supplemented with 10% fetal calf serum fcs, 2 mm glutamine, 05 mgl fungizone and 50 mgl gentamicin. This guide highlights critical steps in the immunofluorescence protocol and demonstrates how protocol changes can. Both cell types were maintained at 37c in the presence of 5% co 2.

The g418resistant transfected clones were constructed after roughly 4 weeks and collected for the downstream experiments. A the viability of h9c2 cells was measured by cck8 assay. An h9c2 21 cell line, originally derived from embryonic left ventricular rat heart tissue through the method of. Cells were either grown in cell culture flasks or seeded into imaging plates from frozen stocks. H9c2 cardiomyoblasts from the american type culture collection atcc, crl1446 rockville, md were cultured in 100mm culture dishes in dulbeccos modi. This can be achieved by using preconfluent cultures cultures that are below their maximum cell density and by changing the culture medium 24 hours before freezing. We show that h9c2 cells are more similar to primary cardiomyocytes than hl1 cells. Protocol example protocol or the culture o the pc3 cell. Ecacc general collection catalogue no 88092904 cell line name. Cell culture and general cell maintenance an h9c2 21 cell line, originally derived from embryonic left ventricular rat heart tissue through the method of selective serial passaging,9 was purchased through sigma aldrich from the european collection of authenticated cell cultures ecacc, salisbury, uk.

We selected the 120 split from a few days previous. What are optimal h9c2 seeding and culture conditions. The h9c2 cell line and primary neonatal cardiomyocyte cells. Undifferentiated h9c2 cells were supplemented daily for a period of 6 12 days, with varying concentrations of retinoic acid ra 10nm, 30nm and 1. H9c2 cell culture and treatment h9c2 cmcs of rat cardiac origin were obtained from guangzhou cellcook biotech co. Cardiomyocyte h9c2 cells exhibit differential sensitivity to. Hek293 hela mcf7 u2 os a549 lncap pc3 hct 116 ht29 hep g2 huh7 k562 jurkat c2c12 raw 264.

H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of alltransretinoic acid ra, creating multinucleated cells with low proliferative capacity. H9c2 cells trouble when culturing h9c2 cells jan172010 hello, im trying to culture the rat heart cell line h9c2 rat heart cells. This line is commonly used in numerous in vitro studies because morphological parameters of their cells resemble immature embryonic cardiomyocytes louch et al. M, in standard cell culture medium containing either 1% foetal bovine, or horse serum, in order to stimulate differentiation of the cells into a more cardiacspecific phenotype.

H9c2 and hl1 cells demonstrate distinct features of energy. The increasing population of nonbeating cells in the culture have different metabolic profiles and have basically lost the only advantage of these cells. The rat cardiomyoblast cell line h9c2 has emerged as a valuable tool for studying. H9c2 cells american type culture collection, manassas, va were maintained in dulbeccos modified eagles medium, without phenol red, supplemented with 0. The study was conducted on an adherent h9c2 line of rat embryonic cardiomyocytes atcc, manassas, va, usa. The rate of cooling may vary but as a general guide a rate of between 1c and 3c per minute will prove suitable for the majority of cell cultures. Immunofluorescence is a powerful tool for elucidating the complex signaling events that underlie biological processes and disease. Alvetex scaffold 6well inserts were prepared for cell culture by washing in 70 % ethanol, 1 x.

Apr 26, 2019 h9c2 cells were transfected with shmalat1 or shnc using lipofectamine 3000 reagent life technologies corporation, carlsbad, ca, usa according to the manufacturers protocol. H9c2 cells, obtained from american type culture collection crl1446. They can be round as well as prolonged ones and it seems to me that the round shape appears. Ac h9c2 cells were subjected to 4 h of ogd followed by reperfusion for 24 h. Cell counting and confluency analysis as quality controls in. Secure a 6cm plate of hek293t cells from the stock incubator. H9c2 cells tissue and cell culture protocol online. An in vitro model of oxidative stress injury was established in cardiac h9c2 cells, and the effects of tanshinone iia were investigated using cell viability, reverse transcription.

H9c2 cell injury was evaluated by measuring b ldh and c ckmb activity. Cultures were grown on dulbeccos modified eagle medium dmem, paa laboratories, pasching, austria, supplemented with 10 % foetal bovine serum life technologies, carlsbad, ca, usa. Erksp53 signal transduction pathway is involved in. Research investigating the aberrant molecular processes that occur during cardiac hypertrophy uses primary cardiomyocytes from neonatal rat hearts as the standard experimental in vitro system. H9c2 cell line is a valuable in vitro model to study the drug. I have been culturing h9c2 for a couple of months in t25 flask and at 70% usually it. Cardiac hypertrophy is a major risk factor for heart failure and associated patient morbidity and mortality. Cell lines successfully transfected using the viafect transfection reagent. H9c2 21 other collection no atcc crl 1446 citation guidance. Wash cells briefly with hanks solution prior trypsin treatment note. Lipofectamine 3000 it has exceeded my expectations and has worked better than our current reagent fugene hd in h9c2 cells. M, in standard cell culture medium containing either 1% foetal bovine, or horse serum, in order to stimulate differentiation of the cells into a more cardiacspecifi c phenotype. H9c2 cells were transfected with shmalat1 or shnc using lipofectamine 3000 reagent life technologies corporation, carlsbad, ca, usa according to the manufacturers protocol. Frozen cells were carefully thawed in a water bath for one min at 37 c, washed once and resuspended in cell culture.

The purpose of this study was to interrogate the mechanism of bnip3 in. Adjust cell seeding and media volumes according to the guidelines provided in the quick start protocol. Recommended transfection protocols for 24well plate. Blood transfusion, red blood cells, metabolites, cytotoxicity, h9c2 cell, hypoxiareoxygenation injury, apoptosis introduction red blood cell rbc transfusion therapy is a common treatment in modern medical prac. This protocol assumes that the cells are being cultured in t150 flasks equipment equipment. Quantitative proteomics and systems analysis of cultured. Cells were resuspended at a concentration of 4 x 10 6 cellsml, 8 x 106 cellsml and 16 x 106 cellsml.

The effect of a number of h9c2 rat cardiomyocytes passage on. H9c2 cells were differentiated in a differentiation medium that comprised dmem supplemented with 1% fbs, ra 10 nm and antibiotics. Rat cardiomyocyte h9c2 cells were obtained from the american type culture collection rockville, md and were grown in dmem containing 10% heatinactivated fbs in an incubator in an atmosphere of 5% co 2, 95% air at 37c. The aim of the present study was to investigate the cardioprotective effect of tanshinone iia and the underlying molecular mechanisms. At the time a cell line is ordered, end users should also consider the culture conditions for the new cell line and make sure the appropriate medium will be available when the cells arrive. Im using dmem high glucose medium 4nm llactate and im having the same problem. Tanshinone iia protects h9c2 cells from oxidative stress. Quickly transfer the ampoule to a 37c water bath until only one or two small ice crystals, if any, remain 12 minutes. H9c2 embryonic rat heartderived ventricular cells myoblasts from atcc were cultured in dulbeccos modified eagles medium dmem, supplemented with 10% fetal bovine serum fbs under 95% air5% co 2, and subcultured when at 5060% confluence. The cell lines listed in table 1 have been transfected successfully by promega corporation.

Cell culture h9c2 cells american type culture collection, manassas, va were cultured in dulbeccos modi. I see big, white, circular shapes at the sides of my flask not moving and in my cell culture floating, im not so sure. The effect of a number of h9c2 rat cardiomyocytes passage. H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. Prepare transfection complexes by mixing 40 l of serum. Using phase contrast microscopy, we th en confirmed that the shapes of the h9c2 cm cells were elongated and partly polynucleated, while the h9c2 cells had spindlelike shapes fig. Altogen biosystems produces preoptimized lipidbased highefficiency transfection reagent kits for the h9c2 embryonic rat myocardium cell line. H9c2 cells trouble hello, im trying to culture the rat heart cell line h9c2 rat heart cells. Cultured cells were grown until they reached subconfluence, followed by detachment with trypsin. In addition, some studies make use of the h9c2 rat cardiomyoblast cell line, which has.

Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. Bcl2 interaction protein 3 bnip3 can be upregulated by hypoxia and participates in the mediation of hypoxiaactivated apoptosis in cardiac myocyte death. May 21, 2018 the handbook and videos provide an introduction to cell culture, with a focus on maintaining cell health throughout the processes of culturing, freezing, thawing and passaging cells. The handbook and videos provide an introduction to cell culture, with a focus on maintaining cell health throughout the processes of culturing, freezing, thawing and. In the present study, we performed for the first time a transcriptional analysis of the h9c2 cell line. This is the first time im working with this cell line, and also the first time this has happened to me using this protocol for subculturing. Cell culture growth conditions recommended transfection reagents plasmid dna transfection and cotransfection. Notably, a higher degree of confluence was associated with lower mitochondrial respiratory activity data not shown. If use of this culture results in a scientific publication, it should be cited in the publication as. Cells from the h9c2 cell line express ach receptors and are often used as transfection hosts in the study of many myocardial diseases. Fa supplementation was performed as previously described 21. Whatever i have gathered based on reading protocols, h9c2 cell needs to be stimulated either by caffeine or using electrodes. Mammalian cell culture 1 introduction to cell culture.

All experiments were carried out before the cells reached 7080% con. Acute myocardial infarction ami results from longterm diminished blood supply diminishment ischemia to the heart, and the main reason for ischemia is hypoxia. Response of h9c2 cardiac cells to palmitate and n3. Transient transfection and development of h9c2 cell lines stably expressing gfpbax, bcl2, and bclx l. The g418resistant transfected clones were constructed after roughly 4. What is the recommended seeding density for h9c2 cells specifically in a plastic tc t25. Blood transfusion, red blood cells, metabolites, cytotoxicity, h9c2 cell, hypoxiareoxygenation injury, apoptosis introduction red blood cell rbc transfusion. Gene expression profiling of h9c2 myoblast differentiation. Pdf the h9c2 cell line and primary neonatal cardiomyocyte cells. H9c2 and hl1 cells demonstrate distinct features of. Original article effects of red blood cell supernatants on.

Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. H9c221 atcc crl1446 rattus norvegicus heartmyocardi. H9c2 rat cardiomyoblast cell line was purchased from the american type culture collection atcc and cultured in mixed growth medium dulbeccos modified eagles medium dmem hyclone supplemented with 10% heatinactivated fbs hyclone. Natural ghrelin and synthetic hexarelin gh secretagogues. Protocol example protocol or the culture o the pc3 cell line. Bax translocates from cytosol to mitochondria in cardiac.