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细胞功能测定

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Staining Methods for cell death

The simplest way: trypan blue. Dead cells stain blue Non-fixed cells: FDA(fluorescein diacetate)-green alive cells; P.I. (propidium iodide)-red dead cells 35 mm plates: to 2 ml medium or PBS add 2 ul ...

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Determination of IC50

DescriptionIn vitro whole cell assay by Hypoxanthine uptake assay Procedure Hypoxanthine uptake assay (with respect to red blood cell cultures of P. falciparumDispense 25 ul complete RPMI in differen ...

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Proliferation Assay: [3H] Thymidine incorporation

Labelling:1) Seed cells in 6 well plates ( in normal growth media) such that there are 5 X 104 cells per well.2) Incubate cells 2 nights.3) Remove media wash cells one time with 1X PBS and add serum f ...

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Cell Clonogenic Survival Assay

DescriptionAllows one to test the capability of adherent cells to survive and replicate following insult with chemicals or radiation. Procedure1. Grow the cells in regular media to 70% confluence and ...

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Timing of Cycles

Materials Monolayer cultures grown in 75 mm culture flasks (Cells from Exercise 11.4 may be used or cultures of tetrahymena yeast or algae may be used.)H-thymidine with at least 4 µc/ml 0.36 c/mMPhosp ...

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Noble Agar Assay

DescriptionCancer cells do not show anchorage and contact inhibition of growth. To assess the anchorage and contact independent growth of cells noble agar assay can be utilized. Noble agar is used to ...

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Pheromone Halo Assay

-Use sterile technique and sterile solutions throughout this method.- 1. Grow a starter culture at 30 C with shaking (250 rpm) until it reaches saturation. ***It is important to make sure all strains ...

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SAPK/Jun kinase assays

Preparation of cell lysate: 1. For cells in suspension grow in DMEM with 10% FCS and then the day before you do the experiment spin the cells down (1 K for 5 min) and obtain a concentration of cells a ...

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T-Cell Activation Using mAb to CD3

Introduction Mature T cells recognize and respond to the antigen/MHC complex through their antigen-specific receptors (TCR). The most immediate consequence of TCR activation is the initiation of signa ...

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Chemotaxis Assay Using Microchemotaxis Chambers (Modified Boyden Chamber)

OverviewThis assay uses a simple apparatus to study chemotaxis of leukocytes or other migratory cells. The apparatus consists of two multi-well chambers separated by a filter containing pores of unif ...

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Isolation and Differentiation of Amnion-Derived Stem Cells (ADSCs)

ADSC isolation culture and cloningRat amnion membrane is mechanically separated from the chorion of embryonic day 18.5 Sprague�Dawley rat embryos The tissue is washed extensively with phosphate-buffer ...

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Intracellular Immunofluorescent Staining for Flow Cytometry

Introduction A modification of the basic immunofluorescent staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular antigens at t ...

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Human Placental Alkaline Phosphatase Stain

The following protocol is for detection of human placental alkaline phosphatase (AP) in cultured cells. Human placental AP is heat stable unlike other AP proteins typically expressed by cells. A few c ...

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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: Trypsinized the cells as for normal passaging until the co ...

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Human Alkaline Phosphatase staining of transgenic embryo/tissue

AP Buffer: 100 mM Tris-HCl pH 9.5 100 mM NaCl 10 mM MgCl2 PTM: PBS 0.1% Tween-20 2 mM MgCl2 Since this is generally done in conjunction with lacZ staining embryo/tissue is usually fixed as per lacZ st ...

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Differentiate ES cells into glial cells and neurons

Day -1: Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells.___________________Day 1: Trypsinized the cells as for normal passaging until the col ...

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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 the cells as for normal passaging until the colonies ...

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MEDIA AND SOLUTIONS REQUIRED FOR ROUTINE ES CELL CULTURE

Media UsedTo prepare 100 ml mediumDMEM 80 ml FCS 15 ml Non-essential amino acids (100x) 1 ml Pen/strep (5000 1U/ml 5000 ug/ml) 1 ml L-Glutamine 200 mM 1 ml Nucleosides stock (100x) 1 ml BME 0.1M 0.2 m ...

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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 1-2 electroporations. Procedure1. Change medium on ...

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ES and TS cell freezing/thawing

Needed:ES cell freezing medium (2x)2x ES cell freezing medium should be made up fresh each time it is to be used and should comprise freshly prepared 60% DMEM+ 20% FCS and 20% DMSO (Sigma Cat. No.D-58 ...

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