细胞功能测定

We use feeder-independent ES cell lines derived from the 129/Ola strain of mice (Nichols et al. Development 110 p.1341 1990). These cells are easy to maintain and significantly reduce the amount of ti ...

IntroductionHuman embryonic stem (hES) cells are pluripotent stem cells derived from pre-implantation embryos that can be maintained and expanded in an undifferentiated state or induced to differentia ...

MEF Conditioned mediumPlate MEFs (p4 or p5) onto gelatin-coated plates at a density of 1x106 cells/10cm culture dish. The next day wash cells with PBS and add 10ml normal hESC medium containing 4ng/ml ...

If culturing in the absence of a feeder cell layer is desired human embryonic stem (hES) cells can be maintained using Mouse or Human-Conditioned Media (Catalog # AR005 AR007). The protocol below has ...

MaterialsTrypsin (Gibco 25200-023)3T3 Medium: 500 mL DME (Invitrogen) + 50 mL FBS (Hyclone) + 5 mL 100x Pen/Strep2x Freezing Medium: 3T3 Medium + 20% DMSO: Filter sterilized.Sterile PBSP100 and P60 ...

Cell are normally passaged every 2-3 days this is important to avoid differentiation.Signs of differentiation are:-i) colonies are surrounded by flattened differentiated cells.ii) large colonies with ...

IntroductionDuring experimental needs the preservation of cell lines and their recovery after preservation are important steps in laboratory. Cryopreservation is a best procedure to preserve the cell ...

You will need: 13.5 day pregnant mouse (we use MTK NEO inbred white mice) 2 sets sterile instruments one containing a pair of curved forceps and a pair of iris scissors one containing two pairs curved ...

Buffers and solutions(All solutions are supplemented with Complete Protease inhibitor tablet (Roche Cat no: 1-873-580) at the final concentration of 1 tablet/50ml):S1 solution: 0.25M sucrose 10 mM MgC ...

LEVEL I Figure 7.3 Isolated microsomes Figure 7.4 TEM of hepatocyte MATERIALS 1% Glutaraldehye (GTA)1% Osmium tetroxideEpoxy or vinyl resin for TEMTEM photomicrograph of liver cellsTransmission electr ...

DescriptionCells are grown to 80% confluency then harvested washed and disrupted in KPM buffer by freezing-thawing cycles with liquid nitrogen essentially as described by Fearnhead et al. 1997 Genes D ...

Flow cell assays are very useful for studying microtubule motility microtubule dynamics kinetochore-microtubule interactions and action of severing/depolymerizing factors on microtubules. Described he ...

LEVEL II Materials Coverslip cultures of an appropriate monolayer cell line Phosphate buffered saline (PBS) Acetone/Methanol (absolute) in a 50:50 volume mixture Rabbit anti-tubulin (or other primary ...

LEVEL III Materials Relaxing Solution0.1 M KCl0.001 M MgCl5 mM ATP0.016 M NaHPONaHPOAdjust pH to 7.30.05 M Sodium phosphate buffer pH 7.00.001 M EDTA (Ethylene diamine tetra acetic acid)BlenderPrepara ...

LEVEL II Materials Freshly removed bovine brain 2Wire sieve (tea strainer)Microtubule buffer (MT buffer)0.1 M MES (2-(N-Morphilino)ethanesulfonic acid)1 mM EGTA (Ethylene Glycol-bis(-aminoethyl Ether) ...

Microtubule spindowns for visual analysis can be performed on single microtubules or microtubules nucleated from axonemes/centrosomes. Although live DIC analysis has largely superseded the use of visu ...

Segmented and polarity-marked microtubules are very useful for many different types of in vitro assays. Segmented microtubules are microtubules with a bright seed and dim elongated segments on both en ...

Negative staining is a rapid qualitative method for analyzing microtubule structure at the EM level. Because negative staining involves deposition of heavy atom stains structural artifacts such as fla ...

Materials Stock Acrylamide: (30%T:0.8%C)30% by weight of acrylamide0.8% by weight of NN'-bis-methylene acrylamideSeparation Gel (Final Concentrations)10% acrylamide (1:3 dilution of stock)0.375 M Tris ...

LEVEL II Materials Coated grid for TEM0.1 M ammonium acetate5% ethanol saturated uranyl acetateTransmission electron microscope Procedure At the conclusion of a 37° C incubation from Exercise 9.6 remo ...