FreezingandThawingcells
Freezing and Thawing cellsFreezingIt is best to freeze cells that are growing rapidly. With adherent cells, it is easiest to set up 100 mm dishes, give them fresh medium the day before you freeze them, and freeze them just as they become confluent. If you are working with suspension cells, make sure that they are growing vigorously before you freeze them. 1. Trypsinize the cells if necessary, and then spin ......阅读全文
Serum－Thawing－－Heat－Inactivation
How to thaw serum: Serum that is stored at -10º C to -40º C is stable for extended periods of time. It is neither necessary or desirable to store se
Thawing－and－Plating－Cryopreserved－Hepatocytes
实验概要This protocol covers thawing and prep of cryopreserved hepatocytes for applications such as metabolic stability, intrinsic clearance, enzyme in
Methods－for－the－Measurement－of－a－Bacterial－Enzyme－Activity－in－Cell－Lysates
Abstract The kinetic characteristics and regulation of aspartate carbamoyltransferase activity were studied in lysates and cell extracts of He
Tissue－Culture－Methods3
REFERENCES:R. Ian Freshney, Culture of Animal cells: A manual of basic techniques, Wiley-Liss, 1987.VI. TISSUE CULTURE PROCEDURESEach student should m
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%
SDS－Whole－Cell－Extracts
-Use sterile technique and sterile solutions in steps 1 to 3.-1. Using a saturated starter culture, inoculate 25 to 30 ml of appropriate media in a 12
Yeast－Genomic－DNA－Prep
Grow 10ml YPD cultures o/n. Figure out cell density; inoculate 30 ml YPD and grow o/n so that cell density is approximately 2 x 108cells/ml the next
The－OP9DL1－System:－Generation－of－TLymphocytes－from－Embryonic4
TROUBLESHOOTINGProblem: The OP9 cells are more than 80%-90% confluent.Solution: It is important when creating working stocks of OP9 cells for freezing
抗生素溶液配制
Working concentrations and stock solutionsPreparation of stock solutionsAmpicillinCarbenicillinGE antibiotic mixTetracyclineChloramphenicol Supplement
Thawing－－Incubating－Human－－Animal－Liver－Microsomes
实验概要BackgroundThe liver is the major organ for metabolism of endogenous substrates as well as exogenous drugs. There are several in vitro tools avai
Master－Cell－Bank
PurposeTo describe the preparation of a Master Cell BankSafetySee SP 09-001 for lab safety considerations for the cell culture lab.EquipmentLaminar Fl
Sauer:Lysing－E.－coli－with－Lysozymes
Getting The Most Out Of Your BugsNative lysis is a staple protocol in practically every biochemistry lab, yet there is significant variability in the
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: Tryp
Differentiating－Neural－Stem－Cells－into－Neurons－and－Glial－Cells
实验概要The protocols in this section describe the steps involved in differentiating neural stem cells (NSC) to neurons, astrocytes, and oligodendrocyte
Cryopreserving－Neural－Stem－Cells
实验概要There are numerous protocols available for cryopreserving neural stem cells (NSCs) derived from human embryonic stem cells; the primary objectiv
Growing－cells
No two cell lines behave exactly the same, so you must learn the peculiarities, or personality, of each of the cell lines with which you work. Irresp
Trypsinizing－cells
There are many procedures with which to trypsinize cells. All include washing the cell monolayer with TD, or in rare cases, with VE. This removes ser
Lyophilizing－Cells
Inoculate 200 ml L-broth supplemented with appropriate antibiotics with the bacteria to be lyophilized. Incubate the culture at 37°C with vig
Lyophilizing－Cells
Lyophilizing Cells Inoculate 200 ml L-broth supplemented with appropriate antibiotics with the bacteria to be lyophilized. Incubate the c
Midiprep－preparation－of－Plasmid－DNA
实验概要The PureLink™ HiPure Plasmid DNA Midiprep Kit allows purification of 100–350 μg of high-quality plasmid DNA from 15–25 mL overnight E. coli cul
Cryopreservation－of－Cell－Lines
AimThe protocol below describes the use of passive methods involving an electric -80ºC freezer for the cryopreservation of cell cultures. ECACC routin
Working－Cell－Bank
1) Keep prepared solutions on ice.2) Determine total cell count of cells to be frozen. (e.g. 1 X 108 )3) Determine number of vials to be frozen. (e.g.
CellTrace™－CFSE－Cell－Proliferation－Kit
实验概要The CellTrace™ CFSE Cell Proliferation Kit provides a versatile and well-retained cell-tracing reagent in a convenient and easy-to-use form. The
TEM－Specimen－Preparation：Preparative－Techniques－for－the－TEM
For routine transmission electron microscopy (TEM), it is generally accepted that specimens should be thin, dry and contain molecules which diffract e
Comparison－of－Enzymatic－and－NonEnzymatic－Means3
MTT Assay on Reattached Cells As seen in Fig. 2 , the proportion of viable MSC that re-attached was significantly higher (p = 0.0004) upon dissoci
Isolation－of－papillary－cells
Isolation of renal papillary cells1. For isolation of papillary cells, kidneys were harvested and kept in HBSS containing 15 mM HEPES, penicillin/
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
Fluorescent－Staining－of－Cells
1. Fluorescent phalloidin in methanol. Phallacidin does not work as well. Dilute 10 ul 330 nM stock into 500 ul PBS for each large coverslip. 2.
KARYOTYPING－ES－CELLS
An actively growing culture of cells is required, i e 2 - 3 d ES cell culture. The total number of cells needs to be between 106 - 107 cells. N B Re
Preparing－cells－and...
实验概要 The method provides a protocol and tips for BrdU staining in tissue sections.Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a syntheti