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 ...

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 plates (Falcon Cat #353046) are coated for 20 to 60 minut ...

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 ...

Media High glucose DMEM (-pyruvate -glutamine)20% Heat-inactivated Fetal calf serum (can vary by cell type be sure!!)1X l-glutamine1X Penicillin/streptomycin1X Non-essential amino acids1X ribonucleosi ...

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 ...

Thawing Cells-Remove a vial of Passage 2 Primary Mouse Embryo Fibroblasts (PMEF'S) at a concentration of 3 x 106 from liquid nitrogen and thaw quickly at 37oC.-Add the 1ml of cell containing media to ...

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 ...

General StrategyWe typically work with tissue culture primary mammalian cells and cell extracts but the protocols can be adapted to other systems such as whole embryos or lower eukaryotes. The cytoske ...

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 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) ...

Materials Siliconized ultracentrifuge microfuge tubes GTP-depleted microtubules 6X SDS loading dye 1X SDS loading dye Coomassie Brilliant Blue R 250 (0.8% in 50% methanol + 10% acetic acid) Destaining ...

Notes:The key variable in MT spindown experiments is ATP. Under high ATP conditionsconventional MAPs are selectively co-sedimented with microtubules. In theabsence of ATP (or in presence of AMPPNP whi ...

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 ...

Determine the OD600 and correlate the cell density from the chart. Set up four 100mL YPD cultures at the following densities: 0.7x105 1x105 and 3.0x105 cells/mL GROWTH OF CELLS Grow 100mL of cells to ...

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 ...

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 ...

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 ...

Materials 3 - 5 Fresh Pig Brains1 M GTP1 M Magnesium SulfatePM buffer = 100 mM Pipes pH 6.9 2 mM EGTA 1 mM Magnesium Sulfate2 mM DTTPM-4M Buffer = 100 mM Pipes pH 6.92 mM EGTA1 mM Magnesium Sulfate2 m ...