Belcher／Knight:ElectrocompetentCells
Electrocompetent CellsFrom Danijela Dukovski at Harvard Medical School. This protocol works well. --Julie NorvilleMaterialsDI water10% GlycerolSpecial EquipmentCentrifugeIce water bathLiquid nitrogenMethodGrow 500ml culture to OD 0.5 (approximately).Spin down cells 5 times in ice cold 10% sterile glycerol.Keep everything on ice and use a refrigerated centrifuge.Each time you resuspend use a progressively smaller volu......阅读全文
Peripheral－blood－“endothelial－progenitor－cells”
EPC Isolation and Characterization1. EPCs were obtained by isolating mononuclear cells using Ficoll density-gradient centrifugation of human blood buf
Isolation－of－human－endometrial－epithelial－cells
Tissue collection1. Endometrial biopsies were collected from women undergoing gynaecological procedures for benign conditions. 2. All women reported
Screen－ES－cells－by－Southern－Blot
Digest DNA in 96-well plateTo each well add:4ul 10Xbuffer4ul Enzyme0.4ul Spermidine(0.4M)31.6ul H2O37‡C 19h, then add 4ul loading dye to each well. Lo
Transfection－of－Mammalian－Cells－Using－Lipofectamine
Materials: LipofectamineBasal Medium containing 10% fetal bovine serum, 1% glutamine, 1% aaBasal Medium containing 1% glutamineBasal Medium cont
Differentiate－ES－cells－into－cardiac－myocytes
Day -1: Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells.____________________Day 1: Trypsini
Differentiate－ES－cells－into－cystic－embryoid－bodies
Day -1: Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells.______________Day 1: Trypsinized th
Preparation－Of－Peripheral－Blood－Cells－For－Chromosome－Analysis
实验概要Lymphocytes are differentiated cells which normally do not undergo subsequent cell divisions. By culturing lymphocytes in the presence of a mito
Isolation－of－human－uterine－smooth－muscle－cells
1. Approval to utilize human tissue was given by Hospital's Committee on Research Involving Human Subjects. 2. Human myometrial and leiomyoma tiss
Tissue－Culture－of－PtK1－cells
Please note: lately (2005 - present), we normally culture PtK1 cells in F-12 media, but they also grow in other types of media as described below.Mixi
Metabolic－Labeling－of－Cells－with－35S
1) Transfer to a 24 wells plate the desired colonies.2) Once the cells are attached (at least 8 hours after tripsinizing them) add ~1 ml ofDME (met-,
Isolation－and－characterization－of－rat－glomerular－endothelial－cells
Glomerular endothelial cells (GECs) from the kidney are in close juxtaposition to other cell types, such as mesangial cells, and may respond to as
Establishment－of－Stable－Transfectant－of－CHO－Lec－Cells
Purpose and BackgroundsCHO lec 3.2.8.1 cellsCHO Lec 3.2.8.1 cells have four independent mutations in the N- and O- glycosylation pathways (Stanley, 19
Cellbased－ELISA－for－primary－cells
1. The 96-well micro-plates are pre-coated with extra cellular matrix.2. Primary cells are grown in the 96-well plates. Seed 100μl of 10,000-20,000
Isolation－of－human－colonic－epithelial－cells－(EC)
1.Human colonic epithelial cells (EC) were isolated from freshly resected colonic surgical specimens obtained. 2.All diagnoses were confirmed by clini
Isolation－of－human－corneal－endothelial－cells－(HCECs)
Isolation of HCECs1. The corneoscleral tissues were rinsed three times with primary cell culture system containing 50 mg/mL gentamicin and 1.25 mg
PriCells:－Isolation－of－endothelial－progenitor－cells－(EPCs)
PriCells: Isolation of endothelial progenitor cells (EPCs) 1. Twenty-four mL venous blood was collected at each time point into Vacutainer CPT Mono
使用饲细胞(Feeder－Cells)制备
（1）取原代培养的人或动物胚胎成纤维细胞，90%汇合时制成细胞悬液，再按105细胞/毫升重新接种培养。（2）在细胞半汇合时，准备0.25μg/ml的丝裂霉素C，按2μg/106细胞的量加入到培养瓶中过夜；或用射线单次照射，剂量30～50戈瑞。（3）细胞经上述处理后，Hanks液漂洗两次、更换培养液、
Isolation－of－human－prostatic－smooth－muscle－cells
Human prostate tissues1.Human hyperplastic prostates were obtained during surgery from four men through transurethral resection of the prostate. 2.All
Isolation－of－peripheral－blood－mononuclear－cells－(PBMC)
1. Acid-citrate dextrose-treated blood (450 ml) was obtained from donors. 2. PBMC were isolated by centrifugation over lymphocyte separation medium. 3
Isolation－and－Culture－of－Human－Liver－Stem－Cells
Materials and Methods1. Human hepatocytes were isolated from fresh surgical specimens of patients undergoing hepatectomies. Healthy liver tissue (
Detection－of－BrdU－Incorporation－in－DNA－Synthesizing－Cells
Detection of BrdU Incorporation in DNA Synthesizing Cells NOTE: Bromodeoxyuridine is a known carcinogen. Propidium iodine (PI) is known to be toxic an
Transient－transfection－into－293T－cells
PurposeTransient transfection into 293T cells is a convenient way to overexpress and obtain both cellular and extracellular (secreted or membrane) pro
Cryopreservation－and－Recovery－of－Mature－Differentiated－Neural－Cells
实验概要Primary neuronal cultures are indispensable in the field of neurobiology and pharmacology. Many researchers favor freshly isolated neuronal cell
Culture－of－human－venous－smooth－muscle－cells
Culture of human venous smooth muscle cells 1. Explants of the 2 groups of skins (control subjects and patients with varicose veins) were prepared
SingleVirus－Tracking－in－Live－Cells
Single-Virus Tracking in Live CellsMichael J. Rust, Melike Lakadamyali, Boerries Brandenburg and Xiaowei Zhuang INTRODUCTIONReal-time, live-cell imagi
Culturing－Rat－Fetal－Neural－Stem－Cells
实验概要Rat neural stem cells (NSCs) serve as a well-established model for investigating human brain development, disease processes, and treatment stra
FACS－Analysis－Using－Peripheral－Blood－Cells
FACS Analysis Using Peripheral Blood CellsCollect blood (75 microliters) into 1ml PBS containing 5 microM EDTA (10 microliters of 0.5 M stock) and mix
ex－vivo－expanded－endothelial－progenitor－cells
Cell Culture. 1. Total hPBMCs were isolated from blood of human volunteers by density gradient centrifugation. 2. Cells were plated on culture dishes
Culture－of－Human－Embryonic－Stem－Cells－(hESC)
All cell lines are initially grown according to the supplier's protocols but we are adapting them to one simple protocol outlined below:6-well pla
Transient－Transfection－of－Cos1－Cells
Transient Transfection of Cos-1 CellsNote: This protocol has been optimized for Cos-1 cells. Successful transfection of each cell type requires optimi