Collagengelcontaining3T3fibroblastsdermalequivalentforraftculture
Collagen gel containing 3T3 fibroblasts (dermal equivalent for raft culture)Ingredients for 6 x collagen matrices in a 6-well plate;Roughly 3x106 J2-3T3s (a fully confluent T75?)1.5mL 10x reconstitution buffer1.5mL 10x DMEM12mL rat tail type 1 collagen (>3.8mg/mL)10N NaOHGlacial acetic acid (in case)MethodPre-chill pipettes, keep collagen on iceThe collagen solidifies above 8ºCMix 1.5mL of 10x DMEM with 1.5mL......阅读全文
Collagen－gel－containing－3T3－fibroblasts－dermal－equivalent－for－raft－culture
Collagen gel containing 3T3 fibroblasts (dermal equivalent for raft culture)Ingredients for 6 x collagen matrices in a 6-well plate;Roughly 3x106 J2-3
Organotypic－raft－culture－of－primary－human－keratinocytes－(PHKs)
Organotypic raft culture of primary human keratinocytes (PHKs)NotesUse 106 keratinocytes per raft.Typical ProtocolTrypsinize the PHKs off the flask an
A－novel－in－vitro－3dimensional－angiogenesis－model
1. Human microvascular endothelial cells (HMVECs) with primary cell kits were cultured on collagen type I-coated dishes to 80% confluency, then ov
Primary－cardiac－fibroblast－and－cardiomyocyte－isolation
1. Ventricles were removed under sterile conditions. 2. Ventricles were placed in cold sterile primary cell culture medium, minced into approximat
Keratocyte－isolation－(corneal－fibroblasts)
Corneal keratocytes (corneal fibroblasts) that this corneal layer is specialized fibroblasts residing in the stroma, representing about 85-90% of
Virus－Infection－of－Fibroblasts
Procedure: Day One 1. Plate cells in 60 mm dishes using 5 ml DMEM containing 10% Calf Serum. The protocol has worked well for NIH3T3 with 1 x 105 cell
Virus－Infection－of－Fibroblasts
Procedure: Day One 1. Plate cells in 60 mm dishes using 5 ml DMEM containing 10% Calf Serum. The protocol has worked well for NIH3T3 with 1 x 105 cell
Primary－Culture－of－Pulmonary－Arterial－Smooth－Muscle－Cells
Primary Culture of Pulmonary Arterial Smooth Muscle Cells 1.Primary cultures of Pulmonary Arterial Smooth Muscle Cells (PASMCs) were isolated from hum
Culturing－Mouse－Embryonic－Fibroblasts
MaterialsTrypsin (Gibco 25200-023)3T3 Medium: 500 mL DME (Invitrogen) + 50 mL FBS (Hyclone) + 5 mL 100x Pen/Strep2x Freezing Medium: 3T3 Medium + 20%
Raft－culture－media－(aka－Green’s－media)
Raft culture media (aka Green’s media)3 parts DMEM (including glutamine or glutamax)1 part Ham’s F125% FCSvarious supplements (not everybody uses the
XC－Assay－of－MoMLV－Virus－Stocks
MaterialsWildtype MoMLV virus aliquot. Stored at -80ºC.Medium: DMEM + 10% FBSNIH 3T3 TK- cellsXC cellsPolybrene 1000x stock = 4 mg/mL, sterile filte
3HThymidine－Uptake－by－Cultured－Cells
Materials Fibroblast cells in log phase growthCa , Mg free-phosphate buffered saline (PBSA)5% (w/v) Glutaraldehyde (GTA)2% (w/v) Perchloric Acid (PCA)
ISOLATION－OF－PRIMARY－MOUSE－EMBRYO－FIBROBLASTS
You will need:13.5 day pregnant mouse (we use MTK NEO inbred white mice)2 sets sterile instrumentsone containing a pair of curved forceps and a pair o
Isolation－of－rat－cardiac－fibroblasts－and－cardiomyocytes
1. Hearts were removed from newborn rats (day 0), put into calcium- and bicarbonate-free HEPES-buffered Hanks’ medium, cut into pieces and digeste
Isolation－and－growth－of－mouse－primary－myoblasts
Isolation of limb muscle from neonatal mice1. Neonatal mice by decapitation or CO2 inhalation.2. Rinse the limbs with 70% ethanol and remove them wi
HThymidine－Uptake－by－Cultured－Cells
H-Thymidine Uptake by Cultured CellsLEVEL IIMaterialsFibroblast cells in log phase growthCa, Mg free-phosphate buffered saline (PBSA)5% (w/v) Glutaral
Analysis－of－Proteins－using－Small－Format－2D－Gel－Electrophoresis
Preparation of protein samplesIntracellular virus proteinsThe following method has been developed principally for the analysis of intracellular protei
Isolation－and－growth－of－pulmonary－artery－adventitial－fibroblasts
1. Adventitia from the main pulmonary artery was harvested neonatal calves. 2. Tissue was collected, carefully dissected free of blood vessels and f
3HThymidine－Uptake－by－Cultured－Cells
3H-Thymidine Uptake by Cultured Cells OverviewA radioactive method for measuring cultured cell growth with 3H-thymidine is described here. MaterialFi
In－vitro－culture－of－embryonic－lungs
In vitro culture of embryonic lungsfrom Hogan LabIsolation of Lung Bud EndodermWhat you need:E11-12 mouse embryosDMEM with 5% fetal bovine serumpetri
InGel－Digestion－of－Proteins－Separated－byPolyacrylamide－Gel－Electrophoresis
1. Excision of protein bands (spots) from polyacrylamide gelsRinse the gloves you use with water to avoid traces of dust in your sample.Rinse the gel
Denaturing－Gradient－Gel－Electrophoresis－(DGGE)
Purpose:Denaturing gradient gels are used to detect non-RFLP polymorphisms. The small (200-700 bp) genomic restriction fragments are run on a low to h
Flow－Sorting－Fibroblasts－with－GFP－and－P.I.
ProtocolWash cells with PBS and trypsinize to a single cell suspension.Count an aliquot on a hemocytometer. Meanwhile centrifuge the cells (e.g. 1000
亲和层析实验技术方法
INTRODUCTIONThis protocol describes a method for removing antibodies that react with bacterially encoded proteins by passing a crude preparation of im
Bacterial－Expression－of－IRS1－containing－GSTfusion－Proteins
1. Grow cells in 5ml (or more) of LB-Amp overnight for a starter culture. 2. Grow larger culture using the overnight culture as a seeding culture.
Exposing－gels－and－plates－containing－radioactive－samples－to－Xray－film
Although most people use the PhosphorImager for western blots, kinase assays and methionine-labeled samples, X-ray film remains the best way to expose
Competitive－RTPCR－Strategy－for－Quantitative－Evaluation－4
We have also been able to detect expression of this receptor in all studied tissues, which is consistent with the pleiotropic nature of growth hormone
Optimized－Fibrin－Gel－Bead－Assay－for－the－Study－of－Angiogenesis
PREPARING CELLSBring up HUVEC and fibroblasts in M199/10% FBS/Pen-Strep (1:100) 1-2 days before beading.Switch medium to EGM-2 (Clonetics) the day bef
关于3T3细胞的基本信息介绍
由Torado等按照严格的传代规律，即每3天在60mm平皿中接种3×105 细胞而获得的小鼠瑞士BAL...由此种细胞系获得的一些克隆，如1-1、1-11、1-13等被用于细胞转化试验，共中以A31-1-13克隆对转化较为敏感。 3T3细胞 3T3 cell 指G．J．Todaro等从Swi
ImmunohistochemistyEnzymatic－Protocol
OverviewR&D Systems provides monoclonal, polyclonal and biotinylated antibodies for immunohistochemical use. The following protocol has been developed