3HThymidineUptakebyCulturedCells
3H-Thymidine Uptake by Cultured Cells OverviewA radioactive method for measuring cultured cell growth with 3H-thymidine is described here. MaterialFibroblast cells in log phase growthCa2+, Mg2+ free-phosphate buffered saline (PBSA)5% (w/v) Glutaraldehyde (GTA)2% (w/v) Perchloric Acid (PCA)Subbed slides (coated with chrom alum gelatin) and PermountNuclear Track Emulsion (Kodak or Ilford)Darkroom and chemica......阅读全文
Subculturing－Adherent－Cells
实验概要The following protocol describes a general procedure for subculturing adherent mammalian cells in culture.主要试剂1. Complete growth medium, pre-warme
Isolation－of－papillary－cells
实验概要This protocols provides a general protocol for isolation of papillary cells.实验步骤Isolation of renal papillary cells1. For isolation of papillary c
Fluorescent－Staining－of－Cells
1. Fluorescent phalloidin in methanol. Phallacidin does not work as well. Dilute 10 ul 330 nM stock into 500 ul PBS for each large coverslip.2. PB
Isolation－of－papillary－cells
Isolation of renal papillary cells1. For isolation of papillary cells, kidneys were harvested and kept in HBSS containing 15 mM HEPES, penicillin/
Collection－of－Peritoneal－Cells
Prepare a 10ml syringe fitted with a 26G short needle and filled with 5 to 7 ml of medium and 2 to 3 ml of air. Air is important.Prepare a Pasteur pip
KARYOTYPING－ES－CELLS
An actively growing culture of cells is required, i e 2 - 3 d ES cell culture. The total number of cells needs to be between 106 - 107 cells.N B Read
Freezing－and－Thawing－cells
Freezing and Thawing cellsFreezingIt is best to freeze cells that are growing rapidly. With adherent cells, it is easiest to set up 100 mm dishes, giv
Transfecting－Suspension－Cells
实验概要将转移基因整合到细胞染色体DNA上,形成稳定表达转移基因的细胞系。 实验原理 细胞转染技术是目前广泛应用于病毒基因结构与功能以及基因调控等的研究。细胞转染可分为短暂转染和稳定(或永久) 转染两种。在短暂转染中,被转染基因并不整合至细胞染色体中,因而不能随细胞分裂而传代。转入病毒基因的转
Electroporation－of－ES－cells
Cells are routinely passaged two days prior to electroporating. Usually one 10 cm plate at approximately 80% confluency will provide enough cells for
Immunofluorescence－Labeling－of－Cells
实验概要Antibodies are an important tool for demonstrating both the presence and the subcellular localization of an antigen. Cell staining is a very ver
Collection－of－Peritoneal－Cells
Prepare a 10ml syringe fitted with a 26G short needle and filled with 5 to 7 ml of medium and 2 to 3 ml of air. Air is important.Prepare a Pasteur pip
The－OP9DL1－System:－Generation－of－TLymphocytes－from－Embryonic2
Freezing ESCs 25. Passage the ESCs as described in Step 22. Resuspend the cells in 3 mL of freezing medium for OP9 cells. 26. Aliquot 1 mL of cell sus
mpulsive－Pressurization－of－Neuronal－Cells－for－Traumatic－Brain－Injury－Study
实验概要A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injur
Isolation－and－culture－of－SpragueDawley－rat－aortic－smooth－muscle－cells
The intact mature arterial media is composed of at least four phenotypically unique cell subpopulations that reside in distinct medial layers. Th
Isolation－of－rodent－pancreatic－β－cells
1. Adult rats weighing 250-350g were anesthetized, sacrificed and immediately used for pancreas sampling.2. Rat islets were isolated from male wistar
Isolation－and－Transcription－Profiling－of－Purified－Uncultured－Human－Strom...
Isolation and Transcription Profiling of Purified Uncultured Human Stromal Stem CellsIsolation and Transcription Profiling of Purified Uncultured Huma
Isolation－and－Culture－of－human－vascular－smooth－muscle－cells
1. Human vascular smooth muscle cells (SMCs) were isolated from human saphenous vein.2. Small moistened pieces of tissue (6 mm2) were placed intima
Reverse－Transfection－for－Gene－Function－Analysis
This guide describes a microarray-based system for the functional analysis in mammalian cells of many genes in parallel. Mammalian cells are cultured
TRIZOL提取注意事项3
3.台式离心机最大能达到2,600×g的离心力，如果将离心时间延长到30-60分钟可以满足步骤2和步骤3中的操作。疑难解答：RNA抽提每1 mg组织或1×106培养细胞预期的RNA产量1.肝和脾，6—10μg2.肾，3—4μg3.骨骼肌和脑组织，1—1.5μg4.胎盘，1-4μg5.上皮细胞(1×1
ELISPOT－(Enzymelinked－ImmunoSPOT)－实验方法步骤1
IntroductionEnzyme-linked immunosorbent spot (ELISPOT) assays were originally developed to enumerate B cells secreting antigen-specific antibodies, bu
Comparison－of－Enzymatic－and－NonEnzymatic－Means1
Comparison of Enzymatic and Non-Enzymatic Means of Dissociating Adherent Monolayers of Mesenchymal Stem CellsThe dissociation of adherent mesenchymal
Human－Embryonic－Stem－(ES)－Cell－Protocols——General－notes－on－ES－cell－culture
hES media has a two week shelf life.hES cells should be cultured in 4, 6, 24, 48, 96 well plates. Growing cells in flasks is not recommended because i
Isolation－of－lymphatic－endothelial－cells
Dermal Cell Suspensions 1. Dermatomed 0.8-mm split-thickness skin was obtained from adult healthy individuals undergoing elective surgery. 2
Transforming－chemically－competent－cells
MethodThaw TSS cells on ice.Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).Note: If you are adding small volumes (~1μl),
FACS－Analysis－of－ES－Cells
Isolate cells and dissociate to single cell suspension (can use Gibco Cell Dissociation Buffer, Accutase or Trypsin)Wash with 10% FBS/DMEM:F12For surf
Culturing－HEK－293－Cells
ReagentsMedium:500 ml Dulbecco’s Modified Eagle Medium (Gibco #41966-029)55 ml FCS (10 %)2.8 ml Gentamycin Solution (Sigma G-1272, 10 ml))TrypsinTryps
Plastic－dishes－for－growing－cells
There are two kinds of dishes used to grow tissue culture cells.Those that are designed for adherent cells have been treated chemically to promote cel
Isolation－of－lymphatic－endothelial－cells
实验概要This protocols provides a general protocol for isolation of lymphatic endothelial cells.实验步骤Dermal Cell Suspensions1. Dermatomed 0.8-mm split-thic
Routine－Splitting－and－freezing－of－cells
1. Grow cells to subconfluence in a flask.2. Harvest as per normal and count.3. Spin down 5min 1.2K in benchtop. Resuspend at 1.0 X 106/ml in 10% DMSO
Freezing－cells－in－liquid－nitrogen
Take off MediaTrypsinate with 1ml x2 Dulbecco A trypsinAdd 7ml MediaPipette up and down to distribute cells throughout media (i.e. not clumped togethe