Protocol 10 Immersion method

If tissues are derived from animals that were not perfused or from human autopsy specimens, put excised tissues organs into fixative for 3-5 h (Bouin's) or overnight (10 neutral-buffered formalin) in intact form, then cut in half to allow for better infiltration of fixative if a large organ (e.g. liver, heart, muscle, spleen). Cutting is generally not required for mouse lymph nodes or thymus because the tissues are small. For brain tissue, fix intact overnight (even if using Bouin's), then cut...

Media and growth conditions

We routinely culture yeast in YPD-rich medium. Once a plasmid is introduced, yeast should be grown in synthetic drop-out medium to select for the plasmid. For example, after transforming YEp51-Bax into yeast, transformants are initially selected and subsequently maintained on synthetic drop-out medium lacking leucine (SD-Leu) and supplemented with glucose to repress Bax expression. We observed that the amount of nutrients in the medium can also affect the sensitivity of yeast to pro-apoptotic...

Immunoblotting procedure

The methods for SDS-PAGE separation and transfer of Bcl-2 family proteins to membranes are not unusual, and thus detailed protocols are not provided here. We have, however, devised an immunoblotting method which permits sequential detection of multiple antigens on a single protein blot without All of these are stored at -20 C in 1 ml aliquots, except aprotinin solution which is stored at 4 C. All of these are stored at -20 C in 1 ml aliquots, except aprotinin solution which is stored at 4 C....

Extraction of sphingosines ceramides and sphingomyelins

Extraction of sphingosines, ceramides, and sphingomyelins is most easily accomplished via two commonly used methods. The most prevalent is the method of Bligh and Dyer (6). Also of use is the Folch extraction (7). The two methods rely upon the hydrophobic organic nature of the sphingolipids to separate them from other cellular components (Figure 3). The resulting Figure 3. Illustration of the phase break induced in the Bligh and Dyer extraction by the addition of water and chloroform. To obtain...

Immunohistochemical detection of Bcl2 family proteins in tissues

The approach we prefer for immunohistochemical detection of Bcl-2 protein is to employ sections derived from paraffin-embedded tissues (Figure 1). This yields morphology which is far superior to cryostat sections and permits use of Figure . Irnmunohistochemical analysis of Bcl-2 family protein in tissue sections. Figure . Irnmunohistochemical analysis of Bcl-2 family protein in tissue sections. Examples are provided of irnmunohistochemical analysis of Scl-2 (A-C). Mcl-1 (D-F), and Bcl-X (G-l)...

General introduction and overview of contents

When cells receive mixed signals for growth they usually die. For instance, when the developmental programme requires cell division but external growth signals are lacking, or when a growth-related gene such as c-myc is highly expressed but the cellular environment has insufficient nutrient content, or a toxic xenobiotic is present, the cell dies by a process termed apoptosis. Although there are differences in the phenomena observed during the apoptotic sequence of events, depending on the cell...

Plasmid considerations

A variety of expression vectors are available to express foreign genes in S. cerevisiae. Two major issues require consideration when choosing a yeast vector for expressing Bax or other Bcl-2 family members in these organisms. First, Bax needs to accumulate to high levels to cause cell death, thus a strong promoter should be employed. Secondly, vectors with appropriate selectable markers should be chosen depending on the yeast strain used. Two general types of promoters are used to achieve high...

Transformation of S cerevisiae by the LiOAc method

Plasmid DNA can be introduced into yeast by several different means. We routinely transform S. cerevisiae using the LiOAc method (74). The transformation efficiency achieved with this method can be influenced by multiple factors. Strain background, cell density at the time of transformation, the quality of the plasmid DNA and carrier DNA used for transformations, and the length of heat-shock can all play some role in influencing the final outcome. When using the protocol below, the...

Chromium release assay

Perhaps the most simple, cheap, and effective technique for in vitro cytotoxicity tests is the one based on target ceil labelling with Na251Cr04. This is probably still the most widely used method and is commonly referred to as 'the chromium (MCr) release assay'. Radioactive chromate passes through the cell membrane by an unknown mechanism and is bound mainly to cyto- plasmic proteins. Since 51Cr is a y-emitter, no special preparation of cell samples is needed for radioactive determination....

Caspase nomenclature and classification

The observation that ced-3, a gene that is essential for apoptosis in the nematode Caenorhabditis elegans, encodes a polypeptide with extensive homology to the cysteine protease interleukin-1 p-converting enzyme (ICE) (8) prompted intensive investigation into the potential role of this family of proteases in various models of apoptosis. To date, 13 mammalian members of this family have been described, 11 in humans (1). A list of the known human caspases, along with their old names, molecular...

Detection of apoptotic cells based on their fractional DNA content

Activation of an endonuclease causes DNA fragmentation (see Chapter 3) and such DNA can be extracted from the cells following their fixation and permeabilization such that less DNA in apoptotic cells stains with any DNA 4 Analysis of ceil death by flow and laser-scanning-cytometry HL-60 CTRL HL-60 CP 3h Annexin V - FITC (log) Annexin V - FITC (log) Annexin V - FITC (log) Annexin V - FITC (log) Figure 4. The distinction between live, early apoptotic, and late apoptotic necrotic cells by LSC...

Morphological recognition of apoptotic cells

POTTEN In this chapter we outline the basic techniques that can be used to identify apoptotic cells on the basis of morphology. All these techniques require the use of some type of microscope, and obviously, the better the microscope then the easier your job is in assessing cell morphology. In addition to the subjective assessment of cellular nuclear appearance, the use of immuno-cytochemical techniques specifically designed for the recognition of apoptotic...

Estimation of target cell nuclear fragmentation using fluorescent dyes

Apoptosis is associated with chromatin condensation and nuclear fragmentation. By using fluorescent dyes that label the DNA, these morphological nuclear changes can be detected by using fluorescence microscopy (see Chapters 2 and 4 for a more detailed discussion). We will describe here briefly a method based on p-phenylenediamine (PPDA) labelling (23), but other fluorescent dyes, like Hoechst 33342, have been successfully used (41) we recommend chapter 2 and the work of Nakajima et al. (50)...

Electron microscopic techniques

Electron microscopy was the original technique used by Kerr et al. (1) in their seminal publication on apoptosis. It provides the most detailed information for the assessment of cell morphology, and hence the most accurate determination of apoptosis in tissues. However, it requires more expensive equipment and takes longer than other methods. Protocol 11 describes a method for transmission electron microscopic study of cell lines and isolated cells from tissues. Prior to starting Protocol 11,...

The annexin V affinity assay

In healthy cells, the phospholipids of the plasma membrane are distributed asymmetrically over the two leaflets of the bilayer. Phosphatidylcholine and sphingomyelin are the predominant species of the outer membrane leaflet and PS is located exclusively in the inner membrane leaflet. This asymmetry is maintained by an aminophospholipid translocase which transports the amino-phospholipids, with preference for PS, from the outer to the inner leaflet (34). In addition, the localization of PS to...

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...

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...

Adjust pH to 7476 with 10 N NaOH 62 Prestaining sample preparation

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

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...