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9.1: Cell Disruption - Biology

9.1: Cell Disruption - Biology


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There are several ways to break open cells.

  • Lysis methods include lowering the ionic strength of the medium cells are kept in. This can cause cells to swell and burst. Mild surfactants may be used to enhance the efficiency of lysis. Most bacteria, yeast, and plant tissues, which have cell walls, are resistant to such osmotic shocks, however, and stronger disruption techniques are often required.
  • Enzymes may be useful in helping to degrade the cell walls. Lysozyme, for example, is very useful for breaking down bacterial walls. Other enzymes commonly employed include cellulase (plants), glycanases, proteases, mannases, and others.
  • Mechanical agitation may be employed in the form of tiny beads that are shaken with a suspension of cells. As the beads bombard the cells at high speed, they break them open. Sonication (20-50 kHz sound waves) provides an alternative method for lysing cells. The method is noisy, however, and generates heat that can be problematic for heat-sensitive compounds.
  • Another means of disrupting cells involves using a “cell bomb". In this method, cells are placed under very high pressure (up to 25,000 psi). When the pressure is released, the rapid pressure change causes dissolved gases in cells to be released as bubbles which, in turn, break open the cells.
  • Cryopulverization is often employed for samples having a tough extracellular matrix, such as connective tissue or seeds. In this technique, tissues are .ash-frozen using liquid nitrogen and then ground to a fine powder before extraction of cell contents with a buffer.

Whatever method is employed, the crude lysates obtained contain all of the molecules in the cell, and thus, must be further processed to separate the molecules into smaller subsets, or fractions.


9.1: Cell Disruption - Biology

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Overview of Cell Lysis and Protein Extraction

All cells have a plasma membrane, a protein-lipid bilayer that forms a barrier separating cell contents from the extracellular environment. Lipids comprising the plasma membrane are amphipathic, having hydrophilic and hydrophobic moieties that associate spontaneously to form a closed bimolecular sheet. Membrane proteins are embedded in the lipid bilayer, held in place by one or more domains spanning the hydrophobic core. In addition, peripheral proteins bind the inner or outer surface of the bilayer through interactions with integral membrane proteins or with polar lipid head groups. The nature of the lipid and protein content varies with cell type and species of organism.

Cell membrane structure. Illustration of a lipid bilayer comprising outer plasma membrane of a cell.

In animal cells, the plasma membrane is the only barrier separating cell contents from the environment, but in plants and bacteria the plasma membrane is also surrounded by a rigid cell wall. Bacterial cell walls are composed of peptidoglycan. Yeast cell walls are composed of two layers of ß-glucan, the inner layer being insoluble to alkaline conditions. Both of these are surrounded by an outer glycoprotein layer rich in the carbohydrate mannan. Plant cell walls consist of multiple layers of cellulose. These types of extracellular barriers confer shape and rigidity to the cells. Plant cell walls are particularly strong, making them very difficult to disrupt mechanically or chemically. Until recently, efficient lysis of yeast cells required mechanical disruption using glass beads, whereas bacterial cell walls are the easiest to break compared to these other cell types. The lack of an extracellular wall in animal cells makes them relatively easy to lyse.

There is no universal protocol for protein sample preparation. Sample preparation protocols must take into account several factors, such as the source of the specimen or sample type, chemical and structural heterogeneity of proteins, the cellular or subcellular location of the protein of interest, the required protein yield (which is dependent on the downstream applications), and the proposed downstream applications. For instance, bodily fluids such as urine or plasma are already more or less homogeneous protein solutions with low enzymatic activity, and only minor manipulation is required to obtain proteins from these samples. Tissue samples, however, require extensive manipulation to break up tissue architecture, control enzymatic activity, and solubilize proteins.

The quality or physical form of the isolated protein is also an important consideration when extracting proteins for certain downstream applications. For instance, applications such as functional enzyme-linked immunosorbent assay (ELISA) or crystallography require not only intact proteins but also proteins that are functionally active or retain their 3D structure.

Examples of protein sources for sample collection. Proteins can come from many sources, including the following: native sources such as mammalian cell cultures, tissues or bodily fluids overexpression in a model system such as bacteria, yeast, insect or mammalian cells monoclonal antibodies from hybridoma cells or plant cells used in agricultural biotechnology.


MATERIALS AND METHODS

Reagents

Restriction enzymes were purchased from Promega (Madison, WI) orNew England Biolabs (Bainbury, MA). Cell culture reagents were purchased from Life Technologies (Grand Island, NY) and fetal bovine serum from Hyclone Laboratories, Inc. (Logan, UT). Enhanced chemiluminescence reagents (ECL) were purchased from Amersham Life Sciences (Arlington Heights, IL). GTPγS was purchased from Boehringer Mannheim (Mannheim, Germany). 9E10 mouse monoclonal antibody (mAb) against a myc peptide (Evan et al., 1985) was diluted from an ascites preparation for most purposes. For immunoelectron microscopy a hybridoma supernatant was used (gift from Dr. R. Parton, European Molecular Biology Laboratory (EMBL)-Heidelberg, Heidelberg, Germany). G1/93 mouse mAb against human ERGIC-53 was a gift from Dr. H.-P. Hauri (Biozentrum, Basel, Switzerland) (Schweizer et al., 1988). Mouse mAbs directed against chicken α-tubulin were purchased from Sigma Chemical Company (St. Louis, MO) or Amersham Life Sciences. Rabbit polyclonal antibodies directed against the 9E10 peptide were a gift from Dr. J. Burkhardt (EMBL-Heielberg). Rabbit polyclonal antibodies directed against bovine GalT and diluted in a bovine serum albumin saline stabilizer solution as a stock solution were a gift from Dr. J. Shaper (Oncology, Johns Hopkins University School of Medicine, Baltimore, MD). Rabbit polyclonal antibodies directed against vesicular stomatitis virus (VSV) G protein were a gift from Dr. K. Simons (EMBL-Heidelberg). Rabbit polyclonal antibodies against mouse IgG and their horseradish peroxidase conjugate were purchased from Sigma Chemical and rabbit polyclonal antibodies against rat Mann-II were purchased from Dr. K. Moremen (University of Georgia, Athens, GA). Ten nanometers protein A-gold were prepared as described (Slot et al., 1991).

Recombinant DNA

The human NAGT-I cDNA (Kumar et al., 1990) with a myc epitope at its extreme carboxyl terminus was prepared by Dr. T. Nilsson in pSRα expression vector (Nilsson et al., 1993) and was a gift from Dr. T. Nilsson (EMBL-Heidelberg). A full-length human ST containing a C-terminal P5D4 VSV epitope (Rabouille et al., 1995a generously provided by Dr. S. Munro, Medical Research Council, Cambridge, United Kingdom) was used as a template for the polymerase cahin reaction to place the 9E10 myc epitope (Evan et al., 1985) at the carboxyl terminus of ST, replacing the P5D4 epitope. This was done so that both transfected glycosyltransferases could be localized with the same antibody. The primers were: 5′ primer, 5′-GGATCCGGATCCCATATGATTCACACCAACCTGAAG-3′, and 3′ primer, 5′-GATCCGGATCCTTACAGGTCTTCTTCAGAGATCAGTTTCTGTTCAGGCAGTGAATGGTCCGGAAGCC-3′. The bases encoding the myc epitope are underlined. The polymerase chain reaction product was digested by BamHI and the resulting DNA was ligated into pSRα at the BamHI site. Recombinant plasmids containing ST-myc with correct orientation with respect to the promoter were selected by restriction enzyme digestion and subsequently confirmed by DNA sequencing.

Cell Culture, Transfection, and Infection with tsO45 VSV

Monolayer Vero (African green monkey kidney cells, ATCC CCL 81) cells were cultured in minimal essentail medium supplemented with 10% heat-inactivated fetal calf serum. Mann-II HeLa cells (a gift from Dr. T. Nilsson, EMBL-Heidelberg Rabouille et al., 1995) were cultured in DMEM supplemented with 10% fetal calf serum and 200 μg/ml Geneticin (G418 sulfate). All cells were routinely grown in 100-mm plastic tissue culture dishes. The cultures were maintained at 37°C in a humidified 5% CO2 incubator.

For DNA transfection, Vero cells were seeded at 1 × 10 6 cells per 100-mm dish 1 d before transfection so that the cells were approximately 70% confluent on the day of transfection. The plasmid DNAs pSRα-NAGT-I-myc or pSRα-ST-myc were purified using CsCl2 gradient centrifugation according to standard methods and dissolved in distilled water at 1 mg/ml final concentration. The calcium phosphate method (Chen and Okyama, 1987) was used for transfection using approximately 20 μg of DNA for each transfection. The precipitate was left in contact with the cells for 16 h. Cells were then rinsed once with PBS and once with calcium- and magnesium-free medium before additional incubation in complete medium. In transient expression experiments, cells were analyzed by indirect immunofluorescence microscopy 24–36 h after transfection. In stable expression experiments, cells were maintained in the above medium containing 600 μg/ml Geneticin for 2 wk before isolating individual clones. Clones were screened by immunofluorescence using 9E10. Almost 100% of the cells were positive for the myc-epitope. ST- and NAGT-I-myc Vero cells were maintained in the presence of 200 μg/ml Geneticin and recultured from frozen stocks about every 4 wk. Expression of NAGT-I-myc was more stable than ST-myc (80% + versus ∼66% positive after 6 wk of continuous culture).

Cells were infected with the ts-O45 VSV and maintained at nonpermissive or permissive conditions for analysis of transport of ts-O45-G protein from the ER as previously described (Storrie et al., 1994). ts-O45 VSV stock was a gift from Dr. K. Simons (EMBL-Heidelberg).

Preparation of Golgi Fractions and Immunoblotting

ST- and NAGT-myc were solubilized from isolated Vero Golgi fractions for immunoblotting. Golgi fractions were prepared by minor variations of the flotation procedure of Balchet al. (1984). In brief, for each preparation, cells were harvested by trypsinization from three-confluent 530-cm 2 Nunc tissue culture trays. Trypsin activity was quenched with complete culture medium. Washed cells were then homogenized on ice in the presence of protease inhibitors by repeated passage through a 25-gauge syringe needle. The total homogenate was then brought to a sucrose concentration of 1.37 M and Golgi was separated from soluble and many membrane components by flotation in a sucrose step gradient in a Beckman SW40 centrifuge rotor (Fullerton, CA) at 4°C. Golgi fractions were collected at the 0.8/1.2 M sucrose interface. The collected fractions were diluted with 0.25 M sucrose containing protease inhibitors and membranous organelles were pelleted by centrifugation in a SW40 rotor at 25,000 rpm for 30 min. Pellets were resuspended in 100 μl of 0.25 M sucrose, quick frozen with liquid N2, and stored at −80°C. Fractions were solubilized by the addition of 3× Laemmli sample buffer (Laemmli, 1970), heated to 95°C, and polypeptides were separated by SDS-PAGE in a Bio-Rad mini-slab gel apparatus (Richmond, CA). Transfer from gel to nitrocellulose was with a Bio-Rad semidry transfer apparatus. Nonspecific binding to the nitrocellullose was blocked with 0.1% Tween 20 and 5% dried milk in PBS. 9E10 was diluted in blocking buffer as was the horseradish peroxidase-conjugated rabbit anti–mouse IgG secondary antibody. ECL reaction product was developed according to the manufacturer’s recommendation. Polypeptide mobility was determined relative to prestained high molecular weight protein standards (Sigma Chemical).

Drug Treatments and Microinjection

Nocodazole was obtained from Sigma Chemical and stored as a 10 mM stock solution in dimethylsulfoxide at −20°C. Immediately before use, stock drug solutions were diluted to final concentration in complete culture medium. Coverslip cultured cells were incubated at 37°C with 10 μM nocodazole or 5 μg/ml BFA (Epicentre Technology, Madison, WI) for various time periods. For drug removal, coverslip cultured cells were transferred three times to fresh dishes containing complete culture medium and then incubated in culture medium supplemented as appropriate at 37°C for various time periods. Cells were treated with cycloheximide (Storrie et al., 1994) and cytochalasin D (Ho et al., 1990) as previously described.

Cells were microinjected with GTPγS, 30 min prior to the addition of nocodazole or shifting ts-O45 VSV-infected cells to permissive temperature using a Zeiss automated injection system (AIS, Carl Zeiss, Jena, Germany). Diluted GTPγS solutions were prepared by diluting aliquots of a 10 mM GTPγS stock solution stored at −20°C to 500 μM in 140 mM KCl and 1 mg/ml aldehyde fixable fluorescein dextran (70 kDa, Molecular Probes, Eugene, OR). Stock GTPγS aliquots were thawed once and used within 1 month of preparation. The injected volume was between 5 and 10% of the total cell volume.

Immunogold Staining of Cryosections and Scoring of Immunogold Distribution across the Golgi Stack

ST- and NAGT-I-myc Vero cells were grown attached to the surface of 100-mm tissue culture dishes in complete medium for 48 h before use. Cells were detached by proteinase K treatment and fixed in 8% formaldehyde as described previously (Griffiths et al., 1984). Ultrathin cryosections were prepared and labeled with antibodies and 10 nm protein A-gold with one significant modification of previously described procedures (Slot et al., 1991) rabbit anti-mouse IgG antibodies were used as bridging antibodies between the mouse monoclonal antibodies and protein A-gold. Quantitation of distribution was performed on Golgi stacks cut perpendicularly to Golgi cisternal profiles in a manner similar to that of Orci et al. (1997) in scoring coatomer, KDEL receptor, and proinsulin distribution. The Golgi was split into three portions for scoring: the outermost cisternae and associated structures facing toward the cell periphery (→ Cell Surface), the medial-Golgi cisterna, and the innermost cisternae and associated structures facing toward the cell nucleus (→ Nucleus). The distribution of single label immunogold particles in association with each portion was scored on micrographs taken at magnifications between 11,800 and 24,700 and printed as full size images on 8- × 10-inch photographic paper.

Immunofluorescence Microscopy

Cells were grown attached to 10-mm round glass coverslips in complete medium for 24 to 48 h before use. After appropriate drug treatment, microinjected cultures were processed in one of two ways: those labeled for localization of Golgi components were fixed with paraformaldehyde and permeabilized with either saponin (Méresseet al. 1995) or 0.1% Triton X-100, whereas those labeled for tubulin distribution were fixed with paraformaldehyde-glutaraldehyde followed by Triton X-100 extraction (see Herzog et al., 1994). In all other situations, cells were transferred directly to −20°C methanol for 4 min (Ho et al., 1990). Double-label antibody combinations were: GalT/myc (9E10), ERGIC-53 (G1/93)/GalT, ERGIC-53 (G1/93)/myc (rabbit polyclonal antibodies), fixable fluorescein isothiocyanate (FITC)-dextran/myc (9E10 or rabbit polyclonal antibodies), and fixable dextran/tubulin. Secondary antibodies raised in donkeys or goats were purchased fromJackson Immunoresearch (West Grove, PA). Non-cross-reactive combinations of FITC, rhodamine, and Texas Red secondary antibodies were used. In some single-label experiments, Cy3-conjugated secondary antibodies were used. Coverslips were mounted in Mowiol. Cells were observed with either a Zeiss IM-35/Axiovert TV100 inverted microscope or a Zeiss Axiophot microscope fitted with a Zeiss planapochromat (63×, numerical aperture 1.40, oil immersion objective). Fluorescein, rhodamine/Cy3, and Texas Red fluorescence were observed with selective Zeiss filter sets. No bleed-through between fluorescence channels was observed. In most experiments, cells were photographed on Kodak TMAX 3200 film for scoring of cytoplasmic Golgi patches and colocalization comparisons. Focal planes for photography were selected to give the maximal number of in-focus scattered, fluorescent Golgi patches.

In some experiments, cells were photographed with either a Photometrics SenSys charge-coupled device (CCD) camera (Photometrics, Phoenix, AZ, 1317 × 1024 pixel Kodak chip) or a Hamamatsu 3-chip color CCD camera (Hamamatsu City, Japan, three 768 × 512 pixel chips). The Photometrics camera was controlled with IPLab Spectrum software (Signal Analytics, Vienna, VA) and the Hamamatsu camera with OpenLab software (Improvision, London, United Kingdom). To correct for pixel shifts resulting from use of separate dichroic mirrors for multilabel fluorescence visualization with the Zeiss IM35 microscope, cells were stained with mixed, differentially conjugated, secondary antibodies to the same primary antibody and photographed with the Photometrics camera. Using the IPLab Spectrum Multiprobe software extension, standard pixel shift corrections were determined averaging observed pixel shifts for a number of different image pairs.

Scoring of Number of Cytoplasmic Fluorescent Patches

Fluorescent micrographs photographed to 35-mm film were printed at an end magnification of 1200×. Images of similar contrast and intensity for each pairing were overlaid with a grid. Golgi protein-positive scattered punctate structures were marked on the grid with a pen and counted excluding the immediate juxtanuclear Golgi complex. For each time point, approximately 30 cells were scored, with a range of 28–33. For each experimental condition, three experiments were performed in the course of establishing the optimal time course for scoring. The overall kinetics appeared identical within each experimental set. Scoring of two representative experiments are shown for GalT distributions providing an indication of data variation (compare A and B in Figure 6 and A and B in Figure 9).


Engineering Fundamentals of Biotechnology

2.56.3.1 Cell Disruption

Cell disruption is required when the product protein is not expressed extracellularly and the host cell has a tough cell wall (i.e., E. coli or yeasts). Several unit operations are available to disrupt the cells and release their contents. Among the most common large-scale cell disruption operations are direct physical disruption methods, including high-pressure homogenization, grinding in ball mills, and cell wall breakage due to ice crystal formation through freeze/thaw of a cell paste. Of these three methods, high-pressure homogenizers offer efficiency, high throughput, and cleanability. These elements make them an attractive platform option, particularly for therapeutic protein production. Chemical or enzymatic lysis approaches also exist, but can be costly both from a raw material perspective and due to the potential need to introduce additional purification operations to remove the lysis additives. Solvent addition can simultaneously disrupt cells and extract the target molecule, as in the case of certain antibiotics.


Conclusion

This study comparing whole genome expression profiling of human and mouse leukocytes has identified for the first time conserved genetic programs common to all LN-DCs or specific to the plasmacytoid versus conventional subsets. In depth studies of these genetic signatures should provide novel insights on the developmental program and the specific functions of LN-DC subsets. The study in the mouse of the novel, cDC-specific genes identified here should accelerate the understanding of the mysteries of the biology of these cells in both mouse and human. This should help to more effectively translate fundamental immunological discoveries in the mouse to applied immunology research aimed at improving human health in multiple disease settings.


Transport in xylem 9.1 HL

This topic relies on a knowledge of the structure of the leaf, its air spaces and stomata. The transport of water through a plant is driven by transpiration in the leaf. Water evaporates from the surfaces of cells and this water vapour fills the air spaces. When stomata are open diffusion takes the water vapour out of the leaf completing the process of transpiration. Xerophytic plants have adaptations to reduce this water loss.

Key concepts

Learn and test your biological vocabulary using these 9.1 Transport in xylem flashcards.

Essentials - quick revision through the whole topic

These slides summarise the essential understanding and skills in this topic.
They contain short explanations in text and images - great revision.

Read the slides and look up any words or details you find difficult to understand.

Exam style question about transport in xylem

Design an experiment to test hypotheses about the effects of abiotic factors on transpiration rates is an important skill from this topic.

Answer the question below on a piece of paper, then check your answer against the model answer below.

The rate of transpiration can be measured using a simple potometer, shown in the diagram. [3]

List three variables that should be controlled to ensure that only wind speed affected the movement of the bubble in the capillary tube. Explain their likely affect on the bubble movement. [3]

Click the + icon to see a model answer.

Model answer

List three variables that should be controlled to ensure that only wind speed affected the movement of the bubble in the capillary tube. Explain their likely affect on the bubble movement. [3]

  • Temperature - if the temperature increases the bubble will move faster, there will be more evaporation of water from the leaf.
  • Health of the plant - if the plant starts to wilt or dies then the bubble may move more slowly because of changes in stomata, or water movement inside the plant.
  • Light intensity - if the light intensity changes this could cause the stomata to open or close, altering the rate of transpiration, and thus the movement of the bubble.
  • Surface area of leaves - it the area of leaves is increased then there will be more air paces for water to evaporate from and the transpiration rate will increase.

Summary list for 9.1 Transport in xylem

Leaf stomata & transpiration

  • Leaves are adapted to absorb carbon dioxide from the air
  • therefore transpiration can also occur in leaves and water is lost to the air.
  • The structure of primary xylem vessels.
  • Xylem vessels transport water from roots to leaves to replace water lost in transpiration.
  • The cohesive and adhesive properties of water molecules allow water transport under tension in xylem and cell walls.
  • The roots cells use active transport for the uptake of mineral ions (nitrates) which causes osmosis and the absorption of water.
  • Xerophytic plants in deserts have adaptations for water conservation.

Adaptations of xerophytes

  • Ability to draw the structure of primary xylem vessels in stems from microscope slides.
  • Recognition of structure and function of xylem. (essential idea)
  • Use potometers to measure transpiration rates
  • Design an experiment to test hypotheses about the effects of abiotic factors on transpiration rates.

Mindmaps

These diagram summaries cover the main sections of topic 9.1 Transport in xylem.
Study them and draw your own list or concept map, from memory if you can.
Try to turn the simple mindmap into the detailed mindmap from memory.

Test yourself - multiple choice questions

This is a self marking quiz containing questions covering the topic outlined above.
Try the questions to check your understanding.

9.1 Transport in xylem 1 / 1

The image on the right shows a stained transverse section of cells in a dicot. leaf magnified about 400x.

Which of the labels in the diagram show the pallisade mesophyl of the leaf?

A: Pallisade mesophyll, B: Spongy mesophyll, C: Air space, D: Guard cell.

The image on the right shows a stained transverse section of cells in a dicot. leaf magnified about 400x.

Which of the labels in the diagram shows the cells which are best adapted to absorb CO2 from air in the air spaces of the lear?

Spongy mesophyll cells are surrounded by air, containing caarbon dioxide.

The image on the right shows a stained transverse section of cells in a dicot. leaf magnified about 400x.

Which of the following sequences best describes the path which oxygen takes within the leaf?

Oxygen is made during photosynthesis in the leaf, and it diffuses into air spaces and then out of the stomata.

The image on the right shows a stained, transverse section of cells in the vein of a dicot. leaf.

It is magnified about 400x.

Which of the labels shows Xylem vessels?

Xylem vessels have no cytoplasm and thick angular walls.
The red cells between A and B and after D are supportive cells surrounding the vascular bundle. Not xylem

Large pale cells with a cytoplasm, a large vacuole and cell walls.

Small cells with thin cell walls

The small elongated cells with a perforated cell wall at their ends

Angular cells with thickened cells walls an no cytoplasm.

Xylem cells are all sizes with thick cell walls, no cytoplasm and angular cell walls. they are found in the lower half of this vascular bundle towards the centre of the stem.

Many of the cells shown in this plant stem are xylem. It is magnified about x400.

Which of the following are adaptations of xylem vessels?

Bands of spiral or ring shaped thickening of cells walls

Absence of nucleus and thin plasma membranes

Many cytoplasmic vesicles and pits in the cell walls

Diagonal cell walls and cytoplasmic streaming

Mature xylem vessels have no cytoplasm. Together they form long tubes
They have thickening of the cell walls to prevent the collapse when water in them is under tention in the transpiration stream.

Which of the following statements explains the importance of adhesion to the transpiration stream?

Adhesion causes air bubbles in xylem vessels.

Adhesion of water to leaf cells helps evaporation of water.

Adhesion between water molecules supports water uptake in the soil.

Adhesion between water molecules and cell walls helps pull water up xylem vessels.

Adhesion is an attraction between water molecules and the inner wall of xylem ducts and the cells of the leaf.

Then water column cannot be pulled away from the wall of xylem ducts due to strong adhesion of water. This helps to create tension, or transpiration pull.

Cohesion and adhesion are properties of water which are important to the transport of water in plants.

Which of the following statements discriminates best between cohesion and adhesion?

Cohesion occurs in xylem vessels but adhesion occurs in other parts of roots and leaves.

Cohesion helps to promote evaporation of water but adhesion reduces evaporation.

Cohesion occurs between water molecules but adhesion is between water molecules and xylem vessels.

Cohesion occurs beetween water molecules and the xylem vessels whereas adhesion is between several water molecules.

Cohesion is the phenomenon of attraction between similar molecules. The water molecules remain attracted by the cohesive force and cannot be easily separated from one another. This force maintains the continuity of the water column in xylem.


Processing Cells for Enzyme Assays

V.J. Cristofalo , Joan Kabakjian , in Tissue Culture , 1973

Explosive Decompression.

Cell disruption by explosive decompression (Parr Bomb, Parr Instrument Co., Moline, Illinois) is based on the fact that nitrogen taken into the cells under high pressure will expand rapidly when the pressure is suddenly reduced to atmospheric levels. The rapid expansion of the gas causes the cells to rupture. For total cell breakage of L-5178Y cells, 11 Manson et al. 12 have described the following procedure. The cell pack is washed into a cellulose nitrate tube with 0.25 M sucrose solution containing 2 × 10 −4 M CaCl2 to a concentration of approximately 5 × 10 8 cells/ml. For total breakage the pressure is raised to 1500 psi and maintained for 15 minutes. At the end of this exposure the cells are allowed to escape through an outlet valve, and the rapid reduction in pressure causes the cells to lyse. We have had only limited experience using this method with diploid cells. A minimum quantity of 5 ml of suspension and, in general, somewhat lower pressures and shorter times are required for breakage of the diploid fibroblastlike cells. A modification of this method appears to be especially useful for the preparation of nuclei as pressure of 500–800 psi for 10 minutes will disrupt the cells but not the nuclei.


Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule

FIG. 1 . Generation of Tslc1 −/− mice. (A) Wild-type allele, targeting construct, and targeted allele of the Tslc1/Igsf4 gene. An open box and solid lines indicate an exon and introns, respectively. IVS1B is a genomic fragment used as a probe for Southern blotting. WT2F and SA7R are the primers for PCR complementary to the wild-type genomic sequence of the Tslc1/Igsf4 gene, while N1F and SA5R are the PCR primers complementary to the targeted allele. P, restriction site of PvuII. (B) Southern blot analysis of the wild-type and targeted alleles of Tslc1/Igsf4. Genomic DNA was digested with a restriction enzyme, PvuII, blotted, and hybridized with a probe, IVS1B. Fragments of 10.9 kb and 2.9 kb were derived from the wild-type and targeted alleles, respectively. (C) PCR analysis for monitoring inheritance of the targeted allele of Tslc1/Igsf4 in the progeny of the Tslc1 +/− intercross. W and T indicate the wild-type allele (1.9 kb) and the targeted allele (1.6 kb), respectively. M, molecular marker. (D) RT-PCR analysis of Tslc1/Igsf4 in the testes from Tslc1 +/+ and Tslc1 −/− mice. A fragment of 154 bp corresponds to exons 1 to 3 of the Tslc1/Igsf4 mRNA. A ribosomal protein gene, S16, served as a control endogenous gene. (E) Western blotting of testis lysates, with or without treatment for deglycosylation, from Tslc1 +/+ , Tslc1 +/ − , and Tslc1 −/− mice. The filter was hybridized with the anti-TSLC1/IGSF4 antibody CC2 (top) or stained with Coomassie brilliant blue (CBB bottom). FIG. 2 . Reproductive organs and semens from Tslc1 +/+ and Tslc1 −/− male mice. (A and B) Morphology of reproductive organs from Tslc1 +/+ (A) and Tslc1 −/− (B) mice. The bladder (open arrow in panel A), prostate (open arrowhead in panel A), seminal vesicles (closed arrowhead in panel A), testes (closed arrow in panels A and B), vasa deferentia (open arrow in B), caput epididymides (closed arrowhead in panel B), and cauda epididymides (open arrowhead in panel B) are demonstrated. Note that the testes from the Tslc1 −/− mice are significantly smaller than those from the Tslc1 +/+ mice. (C to H) Phase-contrast microscopy of semens from Tslc1 +/+ (C and E) and Tslc1 −/− (D and F to H) mice. (I and J) PAS staining of semens from Tslc1 +/+ (I) and Tslc1 −/− mice (J). The open arrowhead indicates a possible acrosome with PAS staining. FIG. 3 . Immunohistochemical and histological analyses of Tslc1 +/+ , Tslc1 +/ − , and Tslc1 −/− mice. (A to H) Immunohistochemical analysis of TSLC1/IGSF4 protein in testes from Tslc1 +/+ (A and D to G), Tslc1 +/ − (B), and Tslc1 −/− (C and H) mice, using the anti-TSLC1/IGSF4 antibodies CC2 (A to F) and EC2 (G and H). (A and B) The TSLC1/IGSF4 protein was detected in the seminiferous tubules but not in the interstitial compartment, including the Leydig cells (open arrowhead in panel A). (C) The TSLC1/IGSF4 protein was not detected in a testis from a Tslc1 −/− mouse. (D) No signals were detected by CC2 preincubated with an excess amount of antigenic polypeptides. (E) Seminiferous epithelium at stage I. The TSLC1/IGSF4 protein was localized along the membranes of step 13 spermatids (closed arrow) and early pachytene spermatocytes (open arrowhead) but was not detected in step 1 spermatids (open arrow) or the Sertoli cells (closed arrowhead). (F) Seminiferous epithelium at stage VII. The TSLC1/IGSF4 protein was localized along the membranes of step 7 spermatids (open arrow), step 16 residual bodies (closed arrow), and preleptotene spermatocytes (closed arrowhead) but was not detected in the late pachytene spermatocytes (open arrowhead). (I to T) Histological analyses of the testes (I to N), ductuli efferentes testis (O and P), epididymides (Q and R), and ovaries (S and T) from Tslc1 +/+ (I, M, O, Q, and S), Tslc1 +/ − (J), and Tslc1 −/− (K, L, N, P, R, and T) mice by HE staining (I to K and M to T) or PAS staining (L). (K) Degenerated round cells were accumulated in the lumen (closed arrowhead), and extensive vacuolization was observed at the basal side (open arrowheads). (L) A large number of round and degenerated cells were seen in the lumen (closed arrowhead). Note that some of the cells in the lumen were stained with PAS and appeared to be derived from round spermatids (open arrowhead), elongating spermatids (open arrow), or the pachytene spermatocytes (closed arrow). (M to R) The open arrows (M and N), open arrowheads (M and Q), and closed arrowheads (N and R) indicate the rete of the testis, the spermatozoa, and the degenerated round cells, respectively. (S and T) Closed arrows indicate the secondary follicle. Mice were examined at 25 weeks of age (A to R) and 40 weeks of age (S and T). FIG. 4 . Staging analyses of the testes from 21-week-old Tslc1 +/+ and Tslc1 −/− mice by PAS staining. FIG. 5 . Flow cytometric analyses of cells isolated from the testes of Tslc1 +/+ and Tslc1 −/− mice. (A) The flow cytograms demonstrate four discrete peaks: an HN (haploid) peak representing elongated spermatids, a 1N (haploid) peak representing round spermatids, a 2N (diploid) peak representing G1-phase spermatogonia, and a 4N (tetraploid) peak representing pachytene spermatocytes and G2-phase spermatogonia. (B) Relative amounts of four cell populations in the testes. Open and closed boxes indicate cells from Tslc1 +/+ and Tslc1 −/− mice, respectively. FIG. 6 . Detection of apoptosis by TUNEL assays. (A and B) Histochemistry of the testes from Tslc1 +/+ (A) and Tslc1 −/− (B) mice by TUNEL assay. Cells stained brown are TUNEL-positive cells. Nuclei were counterstained with methyl green (green). Closed arrowheads and open arrowheads indicate spermatocytes and spermatids, respectively. The open arrow indicates the sloughed cell. (C) Ratios of TUNEL-positive tubules to total tubules. (D) Average numbers of TUNEL-positive cells in TUNEL-positive tubules. FIG. 7 . Morphological analysis of germ cells from Tslc1 +/+ and Tslc1 −/− mice during postnatal development. Testes (T) and epididymides (E) of juvenile mice from 2 to 11 weeks of age were examined by HE staining. Closed arrowheads, closed arrows, open arrowheads, and open arrows in black indicate spermatogonia, spermatocytes, round spermatids, and elongated spermatids, respectively. Closed arrowheads, closed arrows, open arrowheads, and open arrows in yellow indicate spermatozoa, sloughed cells, multinucleated giant cells, and vacuoles, respectively. FIG. 8 . Weights of testes and numbers of normal sperm during postnatal development of Tslc1 +/+ and Tslc1 −/− mice. (A) Weights of testes. (B) Numbers of normal sperm. *, P < 0.05 **, P < 0.005 ***, P < 0.0001. FIG. 9 . Electron microscopic analysis of spermatogenic cells and Sertoli cells from Tslc1 +/+ and Tslc1 −/− mice. (A to H) Spermatids from Tslc1 −/− mice (A to C, G, and H) and Tslc1 +/+ mice (D to F). Spermatids in step 5 (A and D), step 7 (B and E), step 8 (C and F), and step 10 or later (G and H) are demonstrated. (I) A degenerated cell in the epididymis from a Tslc1 −/− mouse. Densely staining materials corresponding to the acrosome (open arrowhead), as well as numerous degenerated vacuoles, were observed. (J to L) Numerous figures of phagocytosis (open arrowhead in panel J) and vacuolization (closed arrowhead in panel J) were observed within the Sertoli cells. Note that the Sertoli cell-Sertoli cell junction (open arrows in panels J and K) and ectoplasmic specialization (closed arrow in panel L) were unaffected in the testes from Tslc1 −/− mice. S, Sertoli cell Sg, spermatogonium Sc, spermatocyte St, spermatid ac, acrosome.


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