Transfer cells to 9 ml warm media in 15ml conical tube. Gently perform pippetting to disperse cells in a cell culture dish, then transfer the suspension to a centrifuge tube. Methods and protocols covers the basic concepts of cancer cell biology and culture while expanding upon the recent shift in cell culture methods from the generation of new cell lines to the use of primary cells. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Mammalian cell tissue culture techniques protocol related view all protocols.
Maintaining healthy cells is the key to experimental success. With ins 1 cells that would be a sub culture ratio of 1. A protocol to enhance ins1e and min6 functionalitythe use of. Free radical modulation of insulin release in ins 1 cells exposed to alloxan. An ins1 rat insulinoma cell line was purchased from the cctcc china center for type culture collection. Rat ins 1 cells were exposed to different concentrations of oxa in vitro and treated with ox1r antagonist sb334867, pi3k antagonist wortmannin, akt. The aim of this study was to develop a protocol to functionalize min6 and ins1e cell lines so they can be used as in vitro models for diabetes research. Our aim is to investigate the role of the aktpkb protein kinase b signaling pathway acting via orexin receptor 1 ox1r and the effects of orexin a oxa on cell proliferation in the insulinsecreting betacell line ins1 cells. Hepatocyte nuclear factor1 modulates pancreatic cell. With the addition of theophylline, we show that min6 and ins1e aggregate cells become responsive to glucose stimulation in a reproducible manner and show stimulation indices 5 and a proper low.
In vitro antidiabetic activity and mechanism of action of. The aim of this study was to investigate the antidiabetic activity and. Procedures cellular biology techniques cell culture animal cell. There is also this paper describing the isolation of an ins 1 derived cell line 832 which my lab though not me personally have experience with. First i must admit that i never cultured ins 1 cells for my experiment by i frequently culture many primary and cancer cell lines. While most cell lines can replicate in more than one culture medium. With ins1 cells that would be a subculture ratio of 1. Perform all activities under aseptic culture conditions. Cell culture media tessem laboratorydiabetes research group. C once this temperature is reached to maintain it here. Feederfree culture conditions are 1 easier to use, 2 more reproducible, and 3 amenable to larger scales.
The most widely used insulinsecreting cell lines are rin, hit, min, ins1 and. Mammalian cell tissue culture techniques protocol abcam. Andrzejewski d, brown ml, ungerleider n, burnside a, schneyer al. Protocol of stable cell line generation creative biomart. The protocal is from thermo fisher scientific important guidelines for transfection 1. Immortal cell lines have been used to analyse protein functions and to examine the effects of exogenous protein expression on cell signalling or transcriptional events. Pancreatic beta cell lines and their applications in. Secure a 6cm plate of hek293t cells from the stock incubator. Ins 1 cells 1 105 cells10cm dish were cultured in medium with or without doxycycline for 24, 48, and 96 h, and cell numbers were counted. Wash cells briefly with hanks solution prior trypsin treatment note. The rat insulinoma cell line ins 1 is a wellestablished model for studies of pancreatic islet beta cell function 1. A protocol to enhance ins1e and min6 functionalitythe use. The cell proliferation reagent wst1 is designed to be used for the nonradioactive, spectrophotometric quantification of cell proliferation, growth, viability, and chemosensitivity in cell populations using the 96wellplate format. Uptodate and highly practical, basic cell culture protocols, third edition, offers basic scientists and clinicianresearchers powerful tools to isolate, culture, and characterize the promising specialized cell types in demand today.
Rat ins1 cells were exposed to different concentrations of oxa in vitro and treated with ox1r antagonist sb334867, pi3k antagonist wortmannin, akt antagonist. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid cluttering 2. Orexin a affects ins1 rat insulinoma cell proliferation via. Simultaneously, we also tried using ripa and several inhibitors cocktails phosphatase inhibitors, and protease inhibitors, then. There is also this paper describing the isolation of an ins1derived cell line 832 which my lab though not me personally have experience with.
The 3t3l1as may be kept in a preadipocyte state by following a standard splitting and feeding protocol. Isolation of ins1derived cell lines with robust atp. For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. Generation of human induced pluripotent stem cells. The extracellular surface of the glp1 receptor is a. Cells were washed with 1x ebss supplemented with 1.
To give you confidence in the health of your cells every step of the way, weve highlighted the technologies and products within cell biology that are critical to maintaining optimal cell. Center for ips cell research and application cira institute for integrated cell material sciences icems kyoto university. G418 concentration, plating density for your specific cell type needs to be determined experimentally. Isolated insulinoma cell lines ins 1, a generous gift of dr. This protocol shows optimum transfection condition using hilymax in neuro2a cells. Ins1 cells were cultured in rpmi 1640 medium gibco, with 11. Cells should only be subcultured when nearly or fully confluent. I was curious about the issue of stalling growth of ins 1 cells in your culture and. In this revised protocol, which we designate protocol version 2, we explain our protocol of human ips cell generation in a practical way focusing on problems we have encountered. Data were normalized using cell number, as determined by manual counting. Pancreatic beta cell lines and their applications in diabetes. Ins1 832 cells cultured in rpmi supplemented with 2 m m glutamine, 1 m m sodium pyruvate, 10% fbs, 10 m m hepes, 100 unitsml penicillin, 100. Experimental protocol for the cell proliferation assay adherent cells grown in microplates 2.
Optimum transfection condition is possibly chaged by passage number and culture condition. Orexin a affects ins1 rat insulinoma cell proliferation. I was curious about the issue of stalling growth of ins 1 cells. Can anyone help me with an ins1 cells growth problem. We used a protocol where it is used 1 pill of sigmafast protease inhibitor in 10 ml of ripa lysis buffer. Human pancreatic beta cell lines culture and insulin assay protocols. Use this background control as a blank position for the elisa reader. Glucagonlike peptide 1 receptor plays a critical role in. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection.
Human pancreatic beta cell lines culture protocols provided. With many recent advances, cancer cell culture research is more important than ever before. Stem cell reports article chemical conversion of human fetal astrocytes into neurons through modulation of multiple signaling pathways jiuchao yin,1,2 lei zhang,1,2 ningxin ma,1 yue wang,1 grace lee,1 xiaoyi hou,1 zhuofan lei,1 fengyu zhang,1 fengping dong,1 gangyi wu,1 and gong chen1, 1department of biology, huck institutes of life sciences, pennsylvania state university. Locate vials in liquid nitrogen and quickly remove with forceps.
Thaw 1 ml aliquot of cells as quickly as possible in water bath at 37oc. Comments in general for initial evaluation, send csf for cell count, glucose also draw simultaneous blood glucose, and protein with gram stain and bacterial culture. In addition, the concern of obtaining mixed cultures of stem cells and fibroblasts is eliminated. It can be used for, the measurement of cell proliferation in response to growth factors, cytokines and nutrients. Every step of the way, a wide range of cell health products maintaining healthy cells is the key to experimental success and reproducible research results. Our aim is to investigate the role of the aktpkb protein kinase b signaling pathway acting via orexin receptor 1 ox1r and the effects of orexin a oxa on cell proliferation in the insulinsecreting beta cell line ins 1 cells. Human pancreatic beta cell lines culture protocols. Take vial to sterile flow hood, gently screw top off slightly to release any pressure. Human pancreatic beta cell lines culture protocols provided by the depositor 1. I was wondering if any of youve tried to check migration of ins1e cells or other beta cells.
I want to use ins1 or ins1e in my research, where can i. Maintenance and generation of seed stocks 1 maintain cultures at a cell concentration between 6 x 103 and 6 x 104 cellscm2. To investigate clonal heterogeneity in ins1 cells while increasing insulin content, the parental cells were stably transfected with the plasmid pcmv8insiresneo containing the human insulin cdna as previously described 17. Spin down and resuspend in appropriate amount of freezing medium. Summary protocol cryopreservation of neurospheres cata sn c to ll f re t. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. Normally closed in order to allow atp synthesis, the ptp leads to mitochondrial depolarization and cell death after extended opening. Cell culture growth conditions,stable cell transfection,transient transfection. Protocol human ips cell culture under feederfree conditions. Ins1 insulin 1, which has been previously assigned to rat chromosome 1, was not linked to these markers. For adults, collect 1020 cc and 1 3 cc for a child for each blood culture set.
Isolated insulinoma cell lines ins1, a generous gift of dr. Ins1 cells 1 105 cells10cm dish were cultured in medium with or without doxycycline for 24, 48, and 96 h, and cell numbers were counted. Stem cell reports article chemical conversion of human fetal astrocytes into neurons through modulation of multiple signaling pathways jiuchao yin, 1,2 lei zhang, 1,2 ningxin ma, 1 yue wang, 1 grace lee, 1 xiaoyi hou, 1 zhuofan lei, 1 fengyu zhang, 1 fengping dong, 1 gangyi wu, 1 and gong chen1. These vessels are seeded with cells, incubated to ensure cell growth and then filled completely with medium for shipping. In south africa, the number of people suffering from diabetes is believed to be rising steadily and the current antidiabetic therapies are frequently reported to have adverse side effects. Human ips cell culture under feederfree conditions. Heidcamp, gustavus adolphus college a large collection of protocols related to cell biology work from microscopy to cell culture, histochemistry, etc. As a guide 1 large confluent flask of the human cell lines can usually be distributed. If so, could you please share some tips or give advice what kind of. Comprehensive and highly practical, plant cell culture protocols, second edition offers todays plant biotechnologists indispensable stateoftheart techniques for carrying out plant research, for developing new transgenic plants, and for todays many and diverse commercial applications. Ins1 cells were nonadherent on cmd and rge but adhered to fibronectin and peptidecoated cmd surfaces and to tissue culture polystyrene tcps. Add required amount of prewarmed cell culture media to fresh flask.
Culturing ins1 cells on cdpgyigsr, rgd and fibronectin. Hepatocyte nuclear factor1 modulates pancreatic cell growth. For ins1 8323 cells were seeded at a density of 3 x 104 cellswell into laminin coated 96well cell culture plates and were incubated for 48 h at 37c in 5% co 2. We selected the 120 split from a few days previous. I cryopreserved these cells a few months ago, but now i just resuscitate them in a six well plate in order to attains a confluency earlier. To give you confidence in the health of your cells every step of the way, weve highlighted the technologies and products within cell biology that are critical to maintaining optimal cell health. Cell growth protocol for panc1 cell line panc1 ucsc genome. Mef plating density there is a wide range of mef plating densities used by researchers for mouse and human pluripotent stem cell culturing. Set up thermometer with a standclamp so that the tip is in the water but not touching the beaker, or else it can melt.
The bdomain, on the other hand, binds an islet cellspecific protein with characteristics similar to those of isl1, a transcriptional activator protein that binds to the e2 enhancer of the rat insulin1 gene. Evaluation of the ins1 832 cell line as a betacell based. Review and cite ins1 protocol, troubleshooting and other methodology information contact experts in ins1 to get answers. Incorporation of 3hthymidine was used as an indicator of ins1. How much time do ins1e and min6 cells need after splitting. Add the same volume of culture medium and cell proliferation reagent wst1 as used in the experiment into one well e. Neuro2a cell optimum transfection condition optimum condition for transfection for 24well plate table 1 optimum condition for tranfection to neuro2a cells cell density serumfree medium 30 l dna 1 g hilymax 2. If an adipocyte phenotype is desired the preadipocytes must be differentiated into adipocytes, a process that takes two weeks following the.
Despite problems associated with beta cell cultures, these cell lines have. Isolation of new ins1derived cell lines with a stable transfection a p p r o a c h. Hilymax neuro2a cell optimum transfection condition. Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. Receipt of frozen cells and starting cell culture 1 immediately place frozen cells in liquid nitrogen storage until ready to culture. Free radical modulation of insulin release in ins1 cells exposed to alloxan. The cells were grown in a humidified atmosphere with 5% co 2 at 37 c. Ethnomedicinal plant use has shown promise for the development of cheaper, costeffective antidiabetic agents with fewer side effects. Our results showed that ins1 832 cells responded conform previous. If cells are less then 7080% confluent but you wish to subculture. Susceptibility to g418 is different among cell lines, which many even vary with cell passage numbers. Ins1e cells migration assay tissue and cell culture. The ins 1 832 cell line is a subclone of ins 1 that was selected for robust gsis, producing and secreting both rat and human insulin.
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