3,206 Works

Electroporation Of Es Cells

Sprotocols
Author: Nagy Lab 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. ### Procedure 1. Change medium on ES cells 3-4 hours prior to electroporation - Gelatinize 10 cm plates, then add 10 ml medium to each. - Place them in a 37 0C incubator until they are required. - Switch on the electroporation apparatus. - Harvest ES cells...

Slide Preparation For Laser Capture Microdissection (Lcm)

Sprotocols
Author: National Cancer Institute *These methods were successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study*. LCM and subsequent molecular analysis can be carried out on slides stained using standard hematoxylin and eosin methods. However, if cell types that are (or are not) expressing a specific protein are required for...

Es / Mef Cell Culture And Electroporation Of Targeting Construct

Sprotocols
Author: Gary Brown ### Day 0 One frozen vial of Murine Embryonic Fibroblasts (MEFs) is thawed quickly in a 37oC water bath. When the last bit of ice is melted, spray the vial with 70% ethanol and transfer the contents of the vial into one 75 cm2 flask (T-75) containing 20 ml of MEF media. Place the MEFs in a 37oC, 5% CO2, 86% humidity incubator. Every frozen MEF preparation thaws a little differently. If...

Processing Of Microdissected Tissue For Molecular Analysis

Sprotocols
Author: National Cancer Institute These methods were successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study. **More than 10,000 Cells** If the amount of microdissected material is substantial (>10,000 cells) then any of the standard procedures for isolating DNA are acceptable. **Less than 10,000 Cells** If the number of cells...

Automated Lineage And Expression Profiling In Live Caenorhabditis Elegans Embryos

Sprotocols
Authors: John Isaac Murray and Zhirong Bao Adapted from [*Imaging in Developmental Biology*](http://www.cshlpress.com/link/imagingdevbiop.htm)(ed. Sharpe and Wong). CSHL Press, Cold Spring Harbor, NY, USA, 2011. ### Abstract Describing gene expression during animal development requires a way to quantitatively measure expression levels with cellular resolution and to describe how expression changes with time. Fluorescent protein reporters make it possible to measure expression dynamics in live cells by time-lapse microscopy, but it can be challenging to identify expressing...

Xenopus Laevis Keller Explants

Sprotocols
Authors: Hazel L. Sive, Robert M. Grainger and Richard M. Harland This protocol was adapted from “Microdissection,” Chapter 10, in [*Early Development of* *Xenopus laevis*](http://www.cshlpress.com/link/xenopus.htm) by Hazel L. Sive, Robert M. Grainger, and Richard M. Harland. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000. ### INTRODUCTION The basic Keller explant is a rectangle of dorsal mesendoderm and ectoderm from an early-gastrula-stage *Xenopus laevis* embryo. It is ~60° to 90° wide, extending from...

Preparing Recombinant Gonad Organ Cultures

Sprotocols
Authors: Blanche Capel and Jordan Batchvarov Corresponding author ([b.capel@cellbio.duke.edu](b.capel@cellbio.duke.edu)) ### INTRODUCTION It can be useful to assay migration between any two adjacent tissues during development. This protocol assays cell migration between the gonad and mesonephros using tissue recombination between genetically marked and unmarked tissue, combined with an organ culture technique. First, agar blocks are prepared in a custom-built mold. The size and shape of the wells are important to maintain the authentic three-dimensional morphology of...

The Genus Antirrhinum (Snapdragon): A Flowering Plant Model For Evolution And Development

Sprotocols
Authors: Andrew Hudson, Joanna Critchley and Yvette Erasmus Corresponding author ([andrew.hudson@ed.ac.uk](andrew.hudson@ed.ac.uk)) ### INTRODUCTION The *Antirrhinum* species group comprises approximately 20 morphologically diverse members that are able to form fertile hybrids. It includes the cultivated snapdragon *Antirrhinum majus*, which has been used as a model for biochemical and developmental genetics for more than 75 yr. The research infrastructure for *A. majus*, together with the interfertility of the species group, allows *Antirrhinum* to be used to examine...

Virus-Induced Gene Silencing As A Tool For Delivery Of Dsrna Into Plants

Sprotocols
Authors: Meenu Padmanabhan and Savithramma P. Dinesh-Kumar1 Corresponding author ([savithramma.dinesh-kumar@yale.edu](mailto:savithramma.dinesh-kumar@yale.edu)) ### INTRODUCTION The inherent RNA silencing mechanism in plants has been effectively manipulated as a tool for the targeted down-regulation of genes. Numerous methods have been employed to initiate this homology-based RNA degradation process, but all rely on the activity of double-stranded RNAs (dsRNAs) corresponding to the gene of interest. Virus-induced gene silencing (VIGS) has gained acceptance as the tool of choice for transient induction...

Xenopus Laevis Keller Explants

Sprotocols
Authors: Hazel L. Sive, Robert M. Grainger and Richard M. Harland This protocol was adapted from “Microdissection,” Chapter 10, in [*Early Development of* *Xenopus laevis*](http://www.cshlpress.com/link/xenopus.htm) by Hazel L. Sive, Robert M. Grainger, and Richard M. Harland. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000. ### INTRODUCTION The basic Keller explant is a rectangle of dorsal mesendoderm and ectoderm from an early-gastrula-stage *Xenopus laevis* embryo. It is ~60° to 90° wide, extending from...

Microinjection Of Dsrna Into Mouse Oocytes And Early Embryos

Sprotocols
Authors: Paula Stein and Petr Svoboda This protocol was adapted from “RNAi in Mouse Oocytes and Early Embryos” contributed by Paula Stein and Petr Svoboda, Chapter 15, in *RNAi: A Guide to Gene Silencing* (ed. Hannon). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003. ### INTRODUCTION This protocol describes how to introduce a double-stranded RNA (dsRNA) of choice into mouse oocytes or fertilized one-cell embryos by microinjection. For collection of mouse oocytes...

Mouse Mutagenesis Using N-Ethyl-N-Nitrosourea (Enu)

Sprotocols
Authors: Andrew P. Salinger and Monica J. Justice1 Corresponding author ([mjustice@bcm.tmc.edu](mjustice@bcm.tmc.edu)) ### INTRODUCTION This protocol describes chemical mutagenesis of male mice using N-ethyl-N-nitrosourea (ENU), which is the most efficient method for obtaining mouse mutations in phenotype-driven screens. A fractionated dose of ENU, an alkylating agent, can produce a mutation rate as high as 1.5 × 10e−3 in male mouse spermatogonial stem cells. Treatment with ENU produces point mutations that provide a unique mutant resource: They...

High-Magnification In Vivo Imaging Of Xenopus Embryos For Cell And Developmental Biology

Sprotocols
Authors: Esther K. Kieserman, Chanjae Lee, Ryan S. Gray, Tae Joo Park and John B. Wallingford Corresponding author ([wallingford@mail.utexas.edu](wallingford@mail.utexas.edu)). ### INTRODUCTION Embryos of the frog *Xenopus laevis* are an ideal model system for in vivo imaging of dynamic biological processes, from the inner workings of individual cells to the reshaping of tissues during embryogenesis. Their externally developing embryos are more amenable to in vivo analysis than internally developing mammalian embryos, and the large size of...

Injection Of Dsrna Into Drosophila Embryos For Rna Interference (Rnai)

Sprotocols
Authors: Leonie Misquitta, Qin Wei and Bruce M. Paterson This protocol was adapted from “Targeted Disruption of Gene Function in Drosophila by RNA Interference,” Chapter 19, in [*Drosophila Protocols*](http://www.cshlpress.com/link/drosprot.htm) (eds. Sullivan et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000. Please note that this version of the protocol is a 2007 revision. ### INTRODUCTION RNA interference (RNAi) is a powerful method for determining the role of specific genes during *Drosophila* embryogenesis....

Fetal Thymus Organ Culture

Sprotocols
Authors: Graham Anderson and Eric J. Jenkinson Corresponding author ([G.Anderson@bham.ac.uk](G.Anderson@bham.ac.uk)) ### INTRODUCTION The generation of functionally competent T-cells from their progenitors involves a series of developmental events including proliferation, differentiation, and survival. T-cell development is a non-cell-autonomous event, and requires interactions with thymic stromal cells. Fetal thymus organ cultures provide an in vitro system in which isolated embryonic thymus lobes can be maintained in culture, allowing the study of T-cell development as well as thymic...

Ex Ovo Electroporation Of Dna Vectors Into Pre-Gastrulation Avian Embryos

Sprotocols
Authors: Cheng Cui, Brenda Rongish, Charles Little and Rusty Lansford Corresponding author ([rusty@caltech.edu](rusty@caltech.edu)) ### INTRODUCTION The transfection of GFP-expressing constructs into early embryos permits key developmental events such as gastrulation to be dynamically imaged using time-lapse video-microscopy. This protocol describes the ex ovo electroporation of a DNA plasmid into avian embryos as young as stage X, nearly 24 h earlier in development than most electroporation protocols. Compared to in ovo electroporation, the ex ovo method...

Fetal Thymus Organ Culture

Sprotocols
Authors: Graham Anderson and Eric J. Jenkinson Corresponding author ([G.Anderson@bham.ac.uk](G.Anderson@bham.ac.uk)) ### INTRODUCTION The generation of functionally competent T-cells from their progenitors involves a series of developmental events including proliferation, differentiation, and survival. T-cell development is a non-cell-autonomous event, and requires interactions with thymic stromal cells. Fetal thymus organ cultures provide an in vitro system in which isolated embryonic thymus lobes can be maintained in culture, allowing the study of T-cell development as well as thymic...

Ex Ovo Electroporation Of Dna Vectors Into Pre-Gastrulation Avian Embryos

Sprotocols
Authors: Cheng Cui, Brenda Rongish, Charles Little and Rusty Lansford Corresponding author ([rusty@caltech.edu](rusty@caltech.edu)) ### INTRODUCTION The transfection of GFP-expressing constructs into early embryos permits key developmental events such as gastrulation to be dynamically imaged using time-lapse video-microscopy. This protocol describes the ex ovo electroporation of a DNA plasmid into avian embryos as young as stage X, nearly 24 h earlier in development than most electroporation protocols. Compared to in ovo electroporation, the ex ovo method...

Dual-Color Elispot Assay For The Simultaneous Detection Of Il-2 And/Or Ifn-Γ Secreting T Cells

Sprotocols
Authors: Salix Boulet, Michel L. Ndongala and Nicole F. Bernard1 Corresponding author ([nicole.bernard@mcgill.ca](nicole.bernard@mcgill.ca)). ### INTRODUCTION The enzyme-linked immunospot (ELISPOT) assay measures the secretion intensity of effector molecules released by immune cells in response to ex vivo antigenic stimulation, as well as the frequency of these responding cells. This assay is highly sensitive, quantitative, easy to use, and amenable to high-throughput screening. For these reasons, the ELISPOT assay is considered by many as a gold standard...

Dual-Color Elispot Assay For The Simultaneous Detection Of Il-2 And/Or Ifn-Γ Secreting T Cells

Sprotocols
Authors: Salix Boulet, Michel L. Ndongala and Nicole F. Bernard1 Corresponding author ([nicole.bernard@mcgill.ca](nicole.bernard@mcgill.ca)). ### INTRODUCTION The enzyme-linked immunospot (ELISPOT) assay measures the secretion intensity of effector molecules released by immune cells in response to ex vivo antigenic stimulation, as well as the frequency of these responding cells. This assay is highly sensitive, quantitative, easy to use, and amenable to high-throughput screening. For these reasons, the ELISPOT assay is considered by many as a gold standard...

Reverse Transfection

Sprotocols
The term reverse transfection comes from the invention and development of a microarray-driven gene expression system by Junald Ziauddin and David M. Sabatini in 2001. As DNA are printed on a glass slide for transfection process to occur before the addition of adherent cells, the order of addition of DNA and adherent cells is a reverse of that of conventional transfection. Hence the word “reverse” is used. ### Reverse Transfection Process **Preparation of transfection mix...

Development Of Mammalian Cell Lines With Lac Operator-Tagged Chromosomes

Sprotocols
Authors: Yuri G. Strukov and Andrew S. Belmont This protocol was adapted from “Development of Mammalian Cell Lines with lac Operator-Tagged Chromosomes,” Chapter 25, in[ *Live Cell Imaging* ](http://www.cshlpress.com/link/livecelp.htm)(eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005. ### INTRODUCTION The discovery and use of fluorescent proteins to label chromosomal proteins has yielded basic structural information as well as insights into dynamics that were previously inaccessible. This protocol describes a...

Design And Cloning Of An Shrna Into A Lentiviral Silencing Vector: Version A

Sprotocols
Authors: Gustavo Tiscornia, Oded Singer and Inder M. Verma This protocol was adapted from “Development of Lentiviral Vectors Expressing siRNA,” Chapter 3, in [*Gene Transfer: Delivery and Expression of DNA and RNA*](http://www.cshlpress.com/link/genetrnp.htm) (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007. ### INTRODUCTION This protocol combines the specificity of small interfering RNA (siRNA)-mediated silencing cassettes with the versatility of lentiviral vectors to stably transduce a wide range of cell...

Collagen Gel Containing 3T3 Fibroblasts (Dermal Equivalent For Raft Culture)

Sprotocols
Author: Matt Lewis ### Ingredients for 6 x collagen matrices in a 6-well plate 1. Roughly 3x10e6 J2-3T3s (a fully confluent T75?) - 1.5mL 10x reconstitution buffer - 1.5mL 10x DMEM - 12mL rat tail type 1 collagen (>3.8mg/mL) - 10N NaOH - Glacial acetic acid (in case) ### Method 1. Pre-chill pipettes, keep collagen on ice - *The collagen solidifies above 8ºC* - Mix 1.5mL of 10x DMEM with 1.5mL of 10x reconstitution buffer,...

Transformation Of Plasmid Dna To Competent E. Coli Cells

Sprotocols
Authors: Chenzhong Kuang ### Material and Reagents 1. SOC - 2% Tryptone - 0.5% Yeast Extract - 10mM NaCl - 10mM MgSO4 - 10mM MgCl2 - 1.5 mL microfuge tubes - 42° C waterbath - Ice - 37° C shaker ### Protocol 1. Thaw competent cells on ice. 20–200µL per tube - Add max. 20µL of a ligation reaction - Mix very gently! - Incubate the tubes on ice for 30 min - Heat shock...

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