Cell Biology Protocols

48 BASIC ELECTRON MICROSCOPY

The dehydrated matrices can be stored overnight, at 4 ◦C. They can then be processed after bringing them to room temperature.

Final sample preparation

Samples are dehydrated by critical point drying from 100% ethanol to air. This must be done to prevent surface tension generated by air-drying from damaging the delicate architecture of filament networks.

17.Critical point drying Transfer the grids to 100% ethanol in the critical point dryer and process according to the apparatus instructions. Store the grids desiccated until they are (optionally) carbon coated.

18.Carbon coating (optional but preferable) When dry, the grids can be viewed in the electron microscope, but the samples are quite delicate and can be vulnerable to beam damage. The specimens can be stabilized by a light coating with carbon in a standard carbon coating apparatus.

Method 2 Resinless sections

This method was designed to observe the structure of the nuclear matrix in cells which have been extracted by the cross-link stabilized nuclear matrix method 1 or the classical nuclear matrix method 1 to reveal the underlying network of branched filaments. It is easily adapted for other, for example cytoskeletal, preparations. Individual proteins can be localized in the nuclear matrix or cytoskeleton using specific antibodies and gold-conjugated second antibodies.

Resinless sections can best be described as a method for infiltrating the delicate structural network of the extracted nucleus with a wax (DGD) to provide support for the fragile structures while they are being sectioned. Thin sections are cut and the

wax is removed, leaving a well-preserved structure without any remaining embedding material.

This method combines the advantages of the whole mount for imaging threedimensional networks with the ability to section. Sectioning is important to image internal nuclear structures that in a whole mount may be obscured by the nuclear lamina. As with whole mount preparations, resinless sections are particularly suitable for the collection of stereo paired images.

Cells are grown in culture on Thermanox coverslips in subconfluent monolayers. They are extracted to reveal the nuclear matrix structure, fixed and processed through all steps in situ while still attached to the coverslip (see Protocol 6.5,

Uncovering the Nuclear Matrix in Cultured Cells). Coverslips are not separated from the cells until after they are embedded with the wax (DGD).

Cultured cells grown in suspension are harvested, extracted and fixed as a small pellet. After the fixation the pellet is placed into a BEEM capsule for further processing. The height of the pellet in the BEEM capsule should be less than 2 mm; if larger, then divide the sample or allow more time for individual steps. 1

1.Coverslips or cell pellets can be processed through all of the same steps (with or without the antibody staining) which have been described above in Method 1. ‘Whole mounts of cells for electron microscopy’, step 1 through post-fixation, step 14.

2.Block staining (optional but preferred) The following staining procedure is not necessary for the ultrastructural visualization of the sample but is very helpful for locating cells in the DGD block prior to sectioning. One of two methods can be selected to stain the cells; both are done at room temperature.

2a. Staining with Delafield’s Hematoxylin. Before beginning cell dehydration, Method 1, step 15, incubate in hematoxylin for 20 min. Continue the dehydration as in Method 1, steps 15 and 16.

or

2b. Staining with Eosin Y. Begin cell dehydration through graded ethanol concentrations, Method 1, step 15, and at the 70% ethanol stage, transfer the samples to a freshly prepared, saturated solution of Eosin Y in 70% ethanol for 20 min. Briefly wash the samples in 70% ethanol and then continue the dehydration as in Method 1, steps 15 and 16.

Transitional solvent

Ethanol and DGD are not miscible so a transition fluid, n-butanol, is used.

3.Transfer the samples to a 1 : 1 v/v mixture of 100% ethanol : n-butanol for 5 min.

4.Transfer cells to 100% n-butanol, two

changes, 5 min each. Put the samples in a 56–60 ◦C oven.

DGD infiltration

The infiltration with DGD is performed at 56–60 ◦C. DGD is melted to this temperature in an oven and samples can be handled in the oven or on the lab bench under an infrared lamp.

5.Prepare a mixture of 1 : 1 v/v DGD : n-butanol and pour it over the samples (use pre-warmed transitional fluid and melted DGD). Leave the samples in the oven, without a cover, for 30 min to allow the transitional solvent to evaporate.

6.Replace the mixture with two changes of pure melted DGD for 30–60 min each change to ensure proper infiltration.

PROTOCOL 2.11

49

7a. For cells on Thermanox coverslips, remove the coverslips from the oven and add a large drop of DGD over the top of the cells. At room temperature, let them cool briefly to solidify. Then, immediately peel the Thermanox coverslips from the solid DGD. Cell structures should come off the coverslip, adhering to the surface of the DGD. Cut squares (about 16 mm2) from the area that contains cells and mount them on DGD stubs with a drop of melted DGD. Trim the blocks for en face EM sectioning.

or

7b. For samples in BEEM capsules (pelleted cells from suspensions), cut away the capsule plastic and the trim the remaining block for EM sectioning.

8.The sectioning is done with glass or diamond knives with troughs filled with distilled water on an ultramicrotome. The sections are collected on carbon coated formvar films supported on copper 200–300 mesh grids. The section thickness, approximately 150 nm, is estimated by the continuous interference colour which is essentially the same as in epon sections.

DGD removal

9.Immerse the grids in n-butanol and incubate them at room temperature. The n-butanol effects a slow extraction and samples are best left for a few hours or overnight.

10.Transfer the grids with sections to a 1 : 1 v/v mixture of 100% ethanol with

the dewaxing solvent n-butanol for

5 minutes

11.Transfer grids to three changes of 100% ethanol, 10 min each.

Final sample preparation

This is the same as in Method 1. Follow the steps for critical point

50 BASIC ELECTRON MICROSCOPY

Drying, Method 1, step 17, and for Carbon Coating, Method 1, step 18.

Note

1 Follow the procedures for the crosslink stabilized nuclear matrix or the classical nuclear matrix methods described in Protocol 6.5, ‘Uncovering the nuclear matrix in cultured cells’.

Acknowledgement

We thank Sheldon Penman, Gabriela Krockmalnic and David Capco, who developed these techniques and passed them along to us.

References

1.Harris, J. R. (ed.) (1991) Electron Microscopy in Biology: A Practical Approach. IRL Press at Oxford University Press, Oxford.

2.Sommerville, J. and U. Scheer (eds) (1987)

Electron Microscopy in Molecular Biology: A Practical Approach, IRL Press, Oxford.

3.Harris, J. R. (1997) Negative Staining and Cryoelectron Microscopy: The Thin Film Techniques. RMS Handbook No. 35, BIOS Scientific Publishers, Oxford.

4.Hajibagheri, M. A. N. (ed.) (1999) Electron Microscopy Methods and Protocols. Humana Press, Totowa, New Jersey, USA.

5.Harris, J. R. and Rickwood, D. (eds) (1999)

Multimedia Methods in Cell Biology, Version 1:0. Chapman & Hall/CRCnetBASE.

6.Harris, J. R. and Scheffler, D. (2001) Routine preparation of air-dried negatively stained and unstained specimens on holey carbon support films: a review of applications. Micron, 33, 461–480.

7.Adrian, M., Dubochet, J., Lepault, J. and McDowall, A. W. (1984) Cryo-electron microscopy of viruses. Nature, 308, 32–36.

8.Adrian, M., Dubochet, J., Fuller, S. D. and Harris, J. R. (1998). Cryo-negative staining. Micron, 29, 145–160.

9.Heuser, J. E. (1983) Procedure for freezedrying molecules adsorbed to mica flakes. J. Mol. Biol., 169, 155–195.

10.Hyatt, A. D. (1991) Immunogold labelling techniques. In: Electron microscopy in Biology: A Practical Approach (J. R. Harris, ed.),

pp. 59–81. IRL Press at Oxford University Press, Oxford.

11.Petry, F. and Harris, J. R. (1999) Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites. Int. J. Parasitol., 29, 1249–1260.

12.Harris, J. R. and Horne, R. W. (1994) Negative staining: a brief assessment of current technical benefits, limitations and future possibilities. Micron, 25, 5–13.

13.Harris, J. R. (1992) Negative staining-carbon film technique: new cellular and molecular applications. J. Elect. Microsc. Tech., 18,

269–276.

14.Reynolds, E. S. (1963) The use of lead citrate

at	high pH as an electron opaque stain
in	electron microscopy. J. Cell Biol., 67,

208–210.

15.Oliver, C. (1994) In: Methods in Molecular Biology Vol. 34: Immunological Methods and Protocols (L. C. Javois, ed.), pp. 291–328. Humana Press, Inc.

16.Bernhard, W. (1969) A new staining procedure for electron microscopical cytology. J. Ultrastruct. Res., 27, 250–265.

17.Biggiogera, M. and Fakan, S. (1998) Fine structural specific visualization of RNA on ultrathin sections. J. Histochem. Cytochem.,

46, 389–395.

18.Penman, S. (1995) Rethinking cell structure.

Proc. Natl Acad. Sci. USA, 92, 5251–5257.

19.Capco, D. G. and Penman, S. (1983) Mitotic architecture of the cell: the filament networks of the nucleus and cytoplasm. J. Cell Biol., 96, 896–906.

20.Capco, D. G., Wan, K. M. and Penman, S. (1982) The nuclear matrix: three-dimensional architecture and protein composition. Cell, 29, 847–858.

21.Capco, D. G., Krockmalnic, G. and Penman, S. (1984) A new method of preparing embedment-free sections for transmission electron microscopy: applications to the cytoskeletal framework and other threedimensional networks. J. Cell Biol., 98, 1878–1885.

22.Nickerson, J. A., Krockmalnic, G., He, D. C. and Penman, S. (1990) Immunolocalization in three dimensions: immunogold staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections. Proc. Nat. Acad. Sci. USA, 87, 2259–2263.

3

Cell Culture

Anne Wilson and John Graham

Protocol 3.1	Tissue disaggregation by mechanical mincing or
	chopping	54
Protocol 3.2	Tissue disaggregation by warm trypsinization	56
Protocol 3.3	Cold trypsinization	58
Protocol 3.4	Disaggregation using collagenase or dispase	60
Protocol 3.5	Recovery of cells from effusions	63
Protocol 3.6	Removal of red blood cells by snap lysis	64
Protocol 3.7	Removal of red blood cells (rbc) and dead cells using
	isopycnic centrifugation	65
Protocol 3.8	Quantitation of cell counts and viability	67
Protocol 3.9	Recovery of cells from monolayer cultures	71
Protocol 3.10	Freezing cells	74
Protocol 3.11	Thawing cells	76
Protocol 3.12	Purification of human PBMCs on a density barrier	80
Protocol 3.13	Purification of human PBMCs using a mixer technique	82
Protocol 3.14	Purification of human PBMCs using a barrier flotation
	technique	83

Cells: isolation and analysis – Anne Wilson

Introduction

The main subcellular components separated from isolated cells include: (i) compact organelles such as mitochondria, nuclei and lysosomes, (ii) membrane systems including the cell membrane, Golgi complex and endoplasmic reticulum, (iii) cytoskeletal elements and (iv) proteins and small cytosolic particles. The four main types of tissue comprise: epithelial, connective, muscle and nervous. All tissues contain specialized cell types which are organized into functional units. The integrity of the tissue is maintained by

Cell Biology Protocols. Edited by J. Robin Harris, John Graham, David Rickwood2006 John Wiley & Sons, Ltd. ISBN: 0-470-84758-1

52 CELL CULTURE

structural and chemical adhesion mechanisms, both between adjacent cells and to the extracellular matrix or basal lamina. Cells may be joined by a variety of specialized junctional complexes involving different cytoskeletal filaments and different families of adhesion molecules, including cadherins and adherins which are often dependent upon divalent cations (Mg++ and Ca++). Dissociation of tissue requires disruption of adhesion with minimal damage to the cells. Since the process of separation may itself compromise cellular integrity and function, awareness of the effect of cell isolation on the viability and function of the intact cells is necessary for optimizing separation conditions for specific cell components.

In turn, the validity of the results obtained from experimental studies on subcellular components is dependent upon the quality of the isolated cells from which those components were derived. Methods used for isolating single cells should maintain viability and metabolic activity of the population; a quantitative baseline to which results can be related and inter-experimental results compared is essential. Determination of cell number, cell viability and protein concentration provides this baseline.

Solid tissues, biological fluids including blood, pleural and abdominal effusions and continuous cell lines are all potential sources for isolated cells. Cells recovered from fresh tissues are likely to be heterogeneous and methods for identifying the isolated cell types are required. Confirmation of cell type derived from cell lines is also advisable.

Heterogeneity of structure between different tissues precludes the use of generic protocols for tissue disaggregation, though features common to tissue-specific protocols include a mechanical means of disaggregating the tissue into small fragments and enzymatic digestion of the supporting matrix for release of individual cells. Variations in enzyme type, enzyme concentrations, exposure time and temperature allow optimization of conditions for different tissues.

A variety of methods described elsewhere [1], for different normal animal tissues are summarized in Table 3.1. Although primarily for setting up cell cultures, they are still relevant in that cell culture requires viable cells with preserved metabolic and reproductive function. Explant cultures are used when the required cell population is represented by only a small number of cells and expansion of numbers is needed.

Rather than offering different protocols for specific tissues, protocols are included that have been used for the disaggregation of human ovarian malignancies. These tumours are sufficiently variable in their characteristics as to require a range of techniques and they provide a basic starting point for mechanical disaggregation and enzyme digestion. The protocols can be followed for different tissue types and used as a baseline for establishing optimal conditions for disaggregation.

Mechanical disaggregation of tissue

Introduction

Mincing or chopping tissue in liquid medium releases cells from the supporting matrix; the isolated cell population often includes large numbers of red blood cells and the viability of nucleated cells may be low as a result of mechanical trauma. However, most disaggregation procedures begin with a mechanical step to reduce the tissue to small fragments, providing a larger surface area for exposure to the digesting enzymes and facilitating penetration of enzymes into the tissue. Soft tissues can be minced finely

	MECHANICAL DISAGGREGATION OF TISSUE	53
Table 3.1 A summary of methods used for disaggregating different tissues [1]
Tissue	Description of disaggregation procedure
Epidermis	Cold trypsinization to separate dermis and epidermis followed by pipetting and
	scraping of separate layers to release keratinocytes
Breast	Recovery of cells from early lactation and post-weaning milk
Cervix	As epidermis or initiation of explant cultures from chopped punch biopsies and
	harvesting of epithelial cells migrating from explants
Liver	Perfusion of blood vessels with a low concentration of collagenase
Pancreas	Digestion with a mix of trypsin and collagenase and filtration to separate acini
	from single cells
Kidney	Digestion of finely chopped kidney with collagenase. Differential sedimentation to
	separate heavier tubules and glomeruli from stromal cells
Bronchus	Growth of epithelial monolayers from bronchial explants using specialized media
	and modified growth surface
Prostate	Digestion of minced tissue with collagenase
Muscle	Digestion of minced tissue with collagenase
Cartilage	Consecutive digestion of chopped fragments with hyaluronidase, then trypsin,
	then collagenase
Bone	Digestion of chopped trabecular bone with collagenase and trypsin
Endothelium	Addition of collagenase to lumen of isolated, intact blood vessel with its free ends
	ligated to retain solution whilst digestion takes place
Brain	Trypsinization of minced tissue

with scissors and harder tissues chopped with opposing scalpel blades. Further release of cells from minced tissue is achieved by vigorous pipetting of the smaller fragments in the suspending fluid, creating shear forces strong enough to separate cells from the fragments.

PROTOCOL 3.1

Tissue disaggregation by mechanical mincing or chopping

Introduction

The biohazards associated with the tissue must be ascertained and appropriate equipment used for handling it [2–5]. In the following protocols, a Class II cabinet is used, protecting both user and tissue from contamination. The practice of aseptic technique is recommended because it ensures that stored media retain sterility and the risk of spurious results from the presence of micro-organisms is minimized. If cells are to be cultured, it is a necessity.

Equipment

All equipment coming into direct contact with tissue must be sterile.

Dissecting scissors (Note: Curved scissors are easier to use in a Petri dish than straight scissors because they can be laid flat along the surface of the dish and thus provide a more extensive cutting surface.)

Forceps

Disposable scalpels

Glass Petri dishes (Note: Dissecting instruments damage plastic dishes and debris may traumatize the cells.)

Pastettes (short-form and long-form)

Universal containers (conical-bottomed) with labels showing volume gradations

Rack for universal containers

Bench centrifuge

Refrigerator

Class II safety cabinet

Reagents

Media coming into direct contact with cells must be sterile.

Hanks balanced salt solution without calcium and magnesium (HBSS) (Note: HBSS is purchased either as a single-strength solution or as a 10 × strength solution. It is more economical to make up singlestrength solution from 10 × strength using tissue culture grade sterile water and adding 5 ml of sterile 7.5% sodium bicarbonate per litre [final concentration of 0.37 g of sodium bicarbonate per litre].)

Storage medium (Note: HBSS has a low nutrient content and, if cells are to be stored for several hours, their viability may be impaired. A variety of culture media are available which can be supplemented with serum to give a semi-defined formulation. More frequently used media include MEM, TC199 and RPMI 1640 [6]. If a fully defined formulation, is required, serum-free media can be used [7].)

Procedure

•Place the tissue into the glass Petri dish and moisten its surface with a small volume of HBSS.

•Dissect away fat, necrotic tissue and connective tissue using a scalpel or scissors and forceps and discard it. (Necrotic tissue is creamy-yellow with a soft, friable texture; it may contain foci of pus.)

•Add a further small volume of HBSS to the tissue and cut it into small fragments.

(Note 1: It is easier to cut or mince the tissue if the volume of liquid is kept small because the fragments then stay on the surface of the dish and cannot float away from the dissecting instruments. Note 2: Mince soft tissue with scissors and chop harder tissue with two opposed scalpel blades.)

•Tilt the dish to an angle of 30◦ from the horizontal and allow the medium and released cells to wash down into the angle of the dish. Use a sterile pastette to aspirate the cell suspension and transfer it to a sterile universal container.

•Add a further small volume of HBSS to the dish with a pastette and wash it over the fragments and surface to release and recover more cells.

•Repeat the last three steps two more times, aiming to reduce the tissue to fragments

PROTOCOL 3.1

55

2 mm across. Pool the released cells from the three mincing/chopping steps.

•Centrifuge the cell suspension at 1000 rpm for 5 min. (Note 1: g = 1.12R (rpm/1000)2, where R = radius of rotor arm from centre of rotor to centre of centrifuge bucket. Note 2: If the final cell suspension is of low viability it may be enhanced by keeping the recov-

ered cells at 4 ◦C and also centrifuging at this temperature. Note 3: Released cells from a mechanically produced suspension may be of low viability because damaged cells separate more easily from the surrounding matrix and the physical forces involved damage cells. However, some tissues are disaggregated effectively with mechanical methods.)

•Decant the supernatant from the cell pellet and resuspend the cells in 10 ml of

storage medium. Store in the refrigerator at 4 ◦C. (Note: The volume of medium

added should be increased to 20 ml if the cell pellet is ≥5 ml.)

•Go to either Protocol 3.8 for cell count and viability determination or to Protocols 3.2–3.4 for enzyme disaggregation.

PROTOCOL 3.2

Tissue disaggregation by warm trypsinization

Introduction

Trypsin has been used for disaggregating tissue for many years [8]. The enzyme damages cells if exposure is prolonged and care must be taken to minimize this when devising tissue-specific protocols. Trypsinization can be carried out at 37 ◦C (warm trypsinization) or at 4 ◦C (cold trypsinization) [9]. DNA released from damaged cells becomes viscous and its presence impairs the recovery of free cells. DNAse is therefore included at low concentrations to digest DNA and reduce viscosity. Serum contains trypsin inhibitors and these must be washed from the tissue before digestion is started. Conversely, the action of trypsin is neutralized by the addition of serum. Under serum-free conditions, soybean trypsin inhibitor can be used instead.

Equipment

All equipment which comes into contact with cells must be sterile.

Wide-necked glass conical flask with a cap made of a double layer of foil extending4 cm down the neck of the flask

Spatula or teaspoon (Note: A teaspoon is convenient for handling larger volumes of tissue.)

Magnetic stirrer bar (Note: Soak in 70% ethanol/30% water and rinse in sterile HBSS to sterilize before use.)

Hotplate/magnetic stirrer (with the heat setting adjusted to maintain a temperature of 37 ◦C or a shaking water bath set at 37 ◦C. (Note 1: Care needs to be taken with a hot plate because of the risk of overheating. To avoid using the heater element the hotplate can be put into an incubator set at 37 ◦C and disaggregation carried out using only the magnetic stirrer. In the absence of a mechanical means of agitating the enzyme/tissue mixture the flask can be incubated at37 ◦C and shaken gently by hand at 5 min intervals. Note 2: A water shaker bath is the most efficient method for maintaining temperature and minimizing mechanical damage during disaggregation.)

Pastettes (long-form and short-form)

Universal containers (conical-bottomed) with a label showing volume gradations

Rack for universal containers

Bijoux

Bench centrifuge

Refrigerator

Freezer (−20 ◦C) for reagent storage

Reagents

All reagents must be sterile.

Trypsin – 2.5% (Note: Thaw out 100 ml of stock solution (2.5%) and store as 10 ml

aliquots in sterile universal containers at −20 ◦C.)

DNAse – 0.4% (Note: Make up stock solution of DNAse Type I in HBSS at

0.4% and filter-sterilize through a sterile

0.22µ filter membrane (eg. Millipore). Store as 1 ml aliquots in sterile bijoux containers at −20 ◦C.)

Hanks balanced salt solution without calcium and magnesium (HBSS)

HBSS or storage medium (see Protocol 3.1) containing 10% serum (e.g. fetal calf or calf) (Note: Soybean trypsin inhibitor can be used instead of serum if serum-free conditions are being used.)

70% methanol/30% water

Procedure

•Thaw out a 10 ml aliquot of 2.5% trypsin and add to 90 ml of HBSS to give a working concentration of 0.25% trypsin. Mix well by swirling the bottle gently. (Note: Do not invert the bottle to mix because it increases the risk of contamination around the neck due to leakage.)

•Thaw out a 1 ml aliquot of DNAse and add to the 100 ml solution of 0.25% trypsin in HBSS to give a working concentration of 0.004%.

•Rinse the minced fragments from mechanical disaggregation in 3 × 10 ml washes of HBSS to remove trypsin inhibitors in the serum and discard the washings. (Note: Viable cells may be recovered from mechanical disaggregation (see Protocol 3.1) but this must be confirmed by cell counts and viability determinations.)

•Transfer the tissue mince from the Petri dish to the conical flask containing a sterile magnetic stirrer bar. (Note: Use the sterile spatula or teaspoon to do this. Use of a wide-necked conical flask makes it easier to drop the fragments to the bottom of the flask without losing them around the neck of the flask.)

PROTOCOL 3.2

57

•Add 10 volumes of 0.25% trypsin/ 0.004% DNAse (pre-warmed to 37 ◦C) to 1 volume of tissue. (Note: 100 ml of solution should be sufficient for three to four trypsinizations of a piece of tissue which was originally about 2 cm3 in volume.)

•Re-cap the flask with the aluminium foil lid and place it on the stirrer. Set

the stir speed	to keep the fragments
in suspension;	there should be a small
vortex with no	frothing.

•Carry out trypsinization for 20–30 min.

•Remove the flask from the stirrer and allow the larger undigested fragments to settle. Remove the foil cap and carefully decant the enzyme solution containing released cells into a universal container. This is most easily done by tilting the flask to allow the fragments to settle into the angle of the flask and using either a long-form pastette or a 10 ml pipette with a bulb to aspirate the cell suspension from the fragments.

•Add an equal volume of serumcontaining medium to the universal to neutralize the effects of the trypsin. (Note: 50 ml centrifuge tubes can be substituted for universals.)

•Centrifuge the universal or centrifuge

tube containing released cells at 1000 rpm for 5 min and resuspend the

cells in medium. Label as T1 and store at 4 ◦C.

•Add fresh, pre-warmed 0.25% trypsin/ 0.004% DNAse to the flask and repeat the last two steps twice more (T2 and T3). (Note: Trypsinization is complete when only fluffy white fragments of connective tissue remain.)

•Go to Protocol 3.8 for cell counting and viability. Determine the viability of the individual harvests and pool those with high cell yield and viability.