Cell lysis and tissue processing procedures 211 Cultured cells
1. a Suspension cells are collected by centrifugation from culture medium, b For adherent cells, remove the culture medium completely from the culture dish. Add 5 ml of PBS and shake gently to wash cells. Then remove PBS completely. Add 5 ml of ice-cold PBS containing PMSF and scrape off with the aid of a rubber policeman do not trypsinize adherent cells . Transfer cells to a 15 ml centrifuge tube and centrifuge at 500 g for 5 min. 2. Remove the supernatant carefully from cell pellets and add...
References
1. Sulston, J.E., Schierenberg, E., White, J.G., and Thomson, J.N. 1983 . Dev. Biol., 100, 64. 2. Wylie, A.H., Morris, R.G., Smith, A.L., and Dunlop, D. 1984 . J. Pathol., 142, 67. 3. Eastman, A., Grant, S., Lock, R., Tritton, T., VanHouten, N., and Yuan, J. 1994 . Cancer Res., 54, 2812. 4. Kerr, J.F.R., Wylie, A.H., and Currie, A.R. 1972 . Br. J. Cancer, 26,239. 5. Skalka, M., Matyasova, J., and Cejkovan, M. 1976 . FEBS Lett., 72,271. 6. Wylie, A.H. 1980 . Nature, 284,555. 7. Oberhamner, F.,...
Filter elution assay to measure apoptotic DNA fragmentation
DNA filter elution is commonly used to study the effects and mechanisms of action of chemotherapeutic drugs and carcinogens 38, 39 . The basic DNA elution filter methods were originally designed to assay DNA damage in intact cells or tissues from living animals 38 . More recently, DNA elution filter assays were adapted to study drug mechanisms in isolated nuclei 40,41 and in a reconstituted cell-free system 13,16 . Altogether, the various elution methods are currently applied in the study of...
Types of DNA fragmentation
DNA fragmentation during apoptosis is considered to occur in two stages. Walker et al. 9 reported sequential degradation of genomic DNA, initially to high molecular weight HMW DNA fragments of approximately 300 kb, followed by the appearance of 50 kb loop-size chromatin fragments which were detected using pulse-field gel electrophoresis. Oligonucleosomal DNA fragments that produce the characteristic DNA ladder are released when the 50 kb fragments are further degraded. Certain cells, such as...
Analysis of sphingoid backbones 41 HPLC on sphingoid backbones
The measurement of sphingoid backbones is most prevalently done by derivat-ization and separation via high performance liquid chromatography HPLC 8 . This method of analysis requires a mild alkaline hydrolysis of the lipids. The sphingoids are then derivatized on their free amine group only available on the sphingoid backbones with ori zo-phthaldialdehyde o-PA , a fluorescent compound. The samples are then injected and separated on HPLC, yielding a peak area for each sphingoid. The sensitivity...
Alkaline hydrolysis of sphingolipids
Analysis of sphingoid backbones and sphingomyelins requires an additional step of refinement. This step is a mild alkaline hydrolysis, which is useful for hydrolysing most glycerolipids, leaving the resistant sphingolipids. Mild alkaline hydrolysis is not used for ceramide measurement when there is additional interest in diacylglycerol, which is sensitive to the procedure. Thus, one can quickly and easily purify the organic lipid extract to one containing mainly sphingolipids. Protocol 3. Mild...
Determination of glutathione levels and its oxidative state
Disruption of critically important oxidants can impair the redox status of the cell, leading to oxidative stress. The oxidative changes reported range from decreased levels of reduced glutathione 23, 24 , -tocopherol 23 , and protein thiols 23 , to downregulation of primary antioxidant defence enzymes such as catalase, manganese superoxide dismutase MnSOD , Cu Zn superoxide dismutase Cu ZnSOD , and thioredoxin 25 . GSH plays a central role in defending cells against radicals and electro-philes...
PCR amplication
1. To a 0.5 ml microcentrifuge, add 8-10 xl of the cDNA reaction mixture from above. 2. Add the following reagents. DEP-treated water, 75-77 xl 10 x reaction buffer Perkin-Elmer Cetus 10 jjlI Taq polymerase 5 units ml , 0.5 xl Final concentration is 25 mM TAPS pH 9.3 , 50 mM KCI, 2 mM MgCI2, 1 mM p-mercaptoethanol. 3. Overlay with 50 xl mineral oil and heat to 94 C for 7 min alternatively, Taq can be omitted until after the heat denaturation, then pipetted under the mineral oil layer into the...
Immunostaining
1. Rinse slides in PBS for 5 min. 2. Incubate for 30 min in 0.03 H202 in PBS. 4. Incubate for 5 min In PBS containing 0.1 glycine. 6. Pre-block and incubate with primary antibodies as described in Protocol 17, steps 1-3. 7. Incubate with secondary antibody differs depending on whether using avidin-biotin-HRPase DAB method described above versus alkaline phosphatase or PAP method for detection see Protocols 20 and 21 . Protocol 20. Alkaline phosphatase method Method 1. Add -0.5 ml of alkaline...
adjust pH to 7476 with 10 N NaOH 62 Prestaining sample preparation
Protocol 12. Deparafinization procedure Method 1. Pre-warm the slides in an oven at 55 C for 1-3 days. This enforces attachment of the tissue to coated slides. If you receive your slides from an outside source, check whether this has already been done. The slides should be placed into a Wheaton glass slide rack for this procedure. Any oven can be used for this. A standard vacuum oven found in most molecular biology laboratories is suitable. We typically hook to house vacuum line and turn on the...
Primers and probes for PCR 541 Mouse bcl2
Forward primer 5'-TGCACCTGAGCGCCTTCAC-3' Reverse primer 5 ' -TAGCTGATTCGACCATTTGCCTGA-3 ' Oligonucleotide internal probe 5'-CCAGGAGAAATCAAACAAAGG-3' expected size of the PCR product is 575 bp Oligonucleotides are endlabelled with 32P -yATP using T4 polynucleotide kinase. Unincorporated 32P ATP is separated from the probe using either ethanol precipitations in the presence of carrier tRNA, DEAE-cellulose minicolurnns Whatman DE-52 , or molecular sieve chromatography centrifuge through Bio-spin 6...
Role of sphingolipids in apoptosis
The role of sphingolipids in the process of apoptosis is centred on the sphingomyelin SM cycle Figure 2 . The inducers of the sphingomyelin cycle include many agents that induce apoptosis and or growth arrest in cells, and examples are cytokines such as TNF-a, interleukin-1, and -y-interferon Fas ligand 1,25-dihydroxyvitamin D3 and environmental stresses such as ultraviolet radiation, serum withdrawal, and chemotherapeutic agents 2 . The initial finding pointing to sphingolipids in apoptosis...
Detection of Bcl2 family proteins by flow cytometry FACS
Bcl-2 family proteins are intracellular antigens and thus their detection by indirect immunofluorescence and subsequent analysis by flow cytometry FACS requires permeabilization of the cells using non-ionic detergents. Procedures are described here specifically for FACS analysis of BcI-2 but are readily adapted to detection of other Bcl-2 family members by adjustment of the choice and amount of primary antibody employed. The immunodetection of Bcl-2 or other Bcl-2 family proteins can be...
Lightscattering properties of apoptotic and necrotic cells
Intersection of a cell with the light of the laser beam in a flow cytometer leads to light scatter, and analysis of the signal of the scattered light reveals information about cell size and structure 34 . The possibility of the analysis of light scattered in the forward and at right angles 'side scatter' 90 directions is a built-in feature of every commercially available flow cytometer utilizing laser illumination, and is a routine measurement, either alone or in conjunction with the...
Analysis of mitochondrial transmembrane potential Aim
Decrease in Avf m is one of the early events of apoptosis 7, 8, 11, see also Chapter 8 . Several types of membrane-permeable lipophilic cationic fluorochromes can serve as probes of Ai gt m in flow or laser-scanning cytometry. When live cells are incubated in their presence the probes accumulate in mitochondria and the extent of their uptake, measured by the intensity of the cellular fluorescence, is considered to reflect Ai m. Rhodamine 123 Rhl23 and 3,3'-dihexiloxa-dicarbocyanine DiOC6 3 are...
In situ detection of DNA strand breaks
In the last few years, techniques based on the detection of DNA strand breaks have gained popularity as methods for the identification of apoptotic cells. Two main techniques exist. They are terminal deoxynucleotide transferase- mediated dUTP-biotin nick-end labelling 16 , usually known by the acronym TUNEL and DNA polymerase l-mediated in situ end-labelling, or ISEL 17, 18 . The incorporation of biotinylated or digoxygenin-iabellcd bases into damaged DNA allows their subsequent detection by...