Cell Biology Protocols

CHEMICAL SAFETY INFORMATION

381

CuSO4	Harmful if swallowed. Irritating to eyes and skin. Do not breathe dust
CuSO4.5H2O	Harmful if swallowed. Irritating to eyes and skin. Eye contact:
	irrigate thoroughly with water for at least 10 min. If discomfort
	persists obtain medical attention. Inhalation: remove from
	exposure, rest and keep warm. In severe cases obtain medical
	attention. Skin contact: wash off skin with water. Remove
	contaminated clothing and wash before reuse. In severe cases,
	obtain medical attention. Ingestion: wash out mouth thoroughly
	with water and give plenty of water to drink. Obtain medical
	attention
Cycloheximide	Very toxic if swallowed, inhaled or absorbed through the skin
Cyclosporin A	Potent immunosuppressant; may cause irritation to eyes, skin,
	mucous membranes. Harmful if swallowed. May cause irritation to
	the eyes, respiratory system and skin
Cytochalasin B	Very toxic by inhalation, in contact with skin and if swallowed
Diaminobenzidine	Carcinogenic
tetrahydrochloride DAB
Diethanolamine	Harmful if swallowed, inhaled or absorbed through skin. Extremely
	destructive of tissues of mucous membranes, upper respiratory
	tract, eyes and skin. Inhalation may be fatal
Diethyl ether (ether)	Extremely flammable: flash point −45 ◦ C. Use in well-ventilated area
	away from sources of ignition. Forms explosive peroxides on
	prolonged storage or on exposure to light. Store only the minimum
	quantity necessary and always use a grade containing a stabilizer
	unless contrary to the chemistry employed in a particular protocol.
	Harmful by inhalation, in contact with skin and if swallowed
Digitonin	Harmful
Dimethyl sulfoxide	Harmful if swallowed. Irritating to eyes and skin. Readily absorbed
(DMSO)	through skin. Avoid breathing vapour. Wear protective clothing.
	May degrade under storage: keep only the minimum quantity
	necessary, under nitrogen if possible
DPX mountant	Contains xylene. Flammable, harmful by inhalation and in contact
	with skin. Irritating to skin. R-phrases: R1O-20/21-38. S-phrases:
	S25-36/37
EDTA	Toxic; irritating to eyes, respiratory system and skin
Eosin	Harmful by inhalation, in contact with skin, and if swallowed.
	Irritating
Ethane	Because of risk of explosion, the reliquification of gaseous ethane,
	taken from a cylinder of liquid ethane, must be carefully
	performed within a negative pressure fume extraction hood. Also,
	following the preparation of specimen grids, the small liquid
	ethane container must be left to evaporate within the fume hood
Ethanol	Highly flammable
	(continued overleaf )

382 SELECTED REFERENCE DATA FOR CELL AND MOLECULAR BIOLOGY

Ethanolamine	Harmful if swallowed, inhaled or absorbed through skin. Material
	extremely destructive of tissues of mucous membranes, upper
	respiratory tract, eyes and skin. Inhalation may be fatal
Ethidium bromide	Harmful. Ethidium bromide is a strong mutagen and can induce
	cancer. Always use gloves and clean up equipment carefully!
Ethyl acetate	Ethyl acetate should be handled with care, and disposed of by means
	appropriate for organic solvents
FITC	Harmful by inhalation, skin absorption or ingestion
Fluorescein isothiocyanate	May be harmful by inhalation, ingestion or skin absorption. Causes
(FITC)	eye and skin irritation. Irritating to mucous membranes and upper
	respiratory tract. Repeated exposure may cause asthma and allergic
	reactions. Prolonged exposure can cause nausea, dizziness,
	headache and lung irritation
Formaldehyde (liquid and	Toxic by inhalation, ingestion and by skin contact (causes
vapour)	sensitization). Possible risk of irreversible effects. Wear gloves and
	eye protection
Freund’s adjuvant	Potentially harmful by inhalation, ingestion or skin absorption. Potent
	inflammatory agent if introduced intradermally or into eyes. In the
	case of skin or eye contact flush with copious amounts of water
	for at least 15 min
Giemsa staining solution	Contains methanol. Highly flammable. Toxic by inhalation and if
	swallowed. R-phrases: R11-23/25. S-phrases: S7-16-24-45
Glutaraldehyde	Glutaraldehyde is harmful if inhaled or if allowed to come into
	contact with the skin. Inhalation of the vapour may cause irritation
	to the mucous membranes, upper respiratory tract, eyes and skin.
	Sensitization with allergic, respiratory and skin reactions may
	occur. Handle in the fume cupboard, and wear gloves
Glycine	May be harmful if swallowed, inhaled or absorbed through skin. May
	cause irritation
Guanidinium	Harmful
hydrochloride
HCl	Very toxic, causes burns
Hexylene glycol	Irritating to eyes and skin
Hydrochloric acid	May be fatal if inhaled, swallowed or absorbed through skin. Causes
	burns. Material extremely destructive of tissues of upper
	respiratory tract, eyes and skin. Inhalation may be fatal
Hydrofluoric acid	Safety precautions must be taken when working with these strong
	acids, particularly hydrofluoric acid, for which an antidote cream
	should be available in case of burns. Hydrofluoric acid must only
	be handled in the fume cupboard, using protective gloves and
	clothing
Hydrogen peroxide	Strong oxidizing agent

CHEMICAL SAFETY INFORMATION

383

125I-iodine	Radioiodine is a γ-emitter and is extremely hazardous. It is essential
	to consult your Radiation Protection Officer for a full list of
	precautions before using it. Minimize the risk of exposure to it by
	working in a designated fume hood with a lead shield between you
	and the source. Minimize also the time of exposure. Wear double
	gloves and lab coat. Prevent aerosol formation and monitor each
	step of the procedure and waste material with a monitor suitable
	for detecting γ-rays

KCl

KH2PO4

KI

Irritating to eyes, respiratory system and skin

Irritating to eyes and skin. Eye contact: irrigate thoroughly with water. If discomfort persists obtain medical attention. Inhalation: remove from exposure. Skin contact: wash off skin with water. Ingestion: wash out mouth thoroughly with water and give plenty of water to drink. In severe cases obtain medical attention

Irritating to eyes and skin. Eye contact: irrigate thoroughly with water. If discomfort persists obtain medical attention. Inhalation: remove from exposure. Skin contact: wash off skin with water. Ingestion: wash out mouth thoroughly with water and give plenty of water to drink. In severe cases obtain medical attention

KNO3	Flammable. Contact with combustible material may cause fire.
	Extinguishing media: carbon dioxide, dry chemical powder or
	appropriate foam
KOH	Causes severe burns (see NaOH for details)
Leishman’s staining	Contains methanol. Highly flammable. Toxic by inhalation and if
solution	swallowed. R-phrases: R11-23/25. S-phrases: S7-16-24-44
Liquid nitrogen	Causes low temperature burns (−196 ◦ C) as liquid and as gas. Wear
	insulated gloves and goggles or face mask. Wear clothing which
	gives good skin and foot protection. Always handle in a
	well-ventilated area to prevent build-up of high nitrogen levels,
	which will reduce the concentration of available oxygen and cause
	asphyxiation
Lysozyme	Harmful
Mayer’s haematoxylin	Harmful and irritating to eyes, respiratory system and skin
Methanol	Used as a fixative and a solvent for stains – highly flammable, toxic
	by inhalation and if swallowed
MgCl2	Irritating to eyes, respiratory system and skin
MgSO4.H2O	Irritating to eyes and skin. Eye contact: irrigate thoroughly with
	water. If discomfort persists obtain medical attention. Inhalation:
	remove from exposure. Skin contact: wash off skin with water.
	Ingestion: wash out mouth thoroughly with water and give plenty
	of water to drink. In severe cases obtain medical attention
	(continued overleaf )

384 SELECTED REFERENCE DATA FOR CELL AND MOLECULAR BIOLOGY

MnSO4.H2O	Harmful: danger of serious damage to health by prolonged exposure
	through inhalation and if swallowed. Do not breathe dust. In case
	of contact with eyes, rinse immediately with plenty of water for
	15 min and seek medical advice. In case of contact, immediately
	wash skin with soap and copious amounts of water. If inhaled,
	remove to fresh air. If not breathing give artificial respiration. If
	breathing is difficult, give oxygen. If swallowed, wash out mouth
	with water provided person is conscious
Na2EDTA.2H2O	Harmful by inhalation, in contact with skin and if swallowed.
	Irritating to eyes, respiratory system and skin. In case of contact
	with eyes, rinse immediately with plenty of water for 15 min and
	seek medical advice.

Na2HPO4

Na2MoO4.2H2O

Irritating to eyes, respiratory system and skin

Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. In case of contact with eyes, rinse immediately with plenty of water for 15 min and seek medical advice. In case of contact, immediately take off all contaminated clothing and wash skin with soap and copious amounts of water. If inhaled, remove to fresh air. If not breathing give artificial respiration. If breathing is difficult, give oxygen. If swallowed, wash out mouth with water provided person is conscious

NaOH	Very corrosive, causes severe burns. Eye contact: rinse immediately
	with plenty of water for 15 min and seek medical advice. Skin
	contact: immediately wash skin with soap and copious amounts of
	water. Ingestion: if the chemical has been confined to the mouth
	give large quantities of water as a mouthwash. Ensure the mouth
	wash is not swallowed. If the chemical has been swallowed, give
	about 250 ml of water to dilute it in the stomach. In severe cases,
	obtain medical attention

N -ethylmaleimide (NEM)

NH4NO3

Toxic; irritating to eyes, respiratory system and skin

Flammable: Contact with combustible material may cause fire. Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. In case of contact with eyes, rinse immediately with plenty of water for 15 min and seek medical advice. In case of contact, immediately take off all contaminated clothing and wash skin with soap and copious amounts of water. If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. If swallowed, wash out mouth with water provided person is conscious

Nicotinic acid	Avoid contact with skin and eyes
Nitric acid	Corrosive. Safety precautions must be taken when working with
	strong acids. Nitric acid must only be handled in the fume
	cupboard, using protective gloves and clothing

CHEMICAL SAFETY INFORMATION

385

o-phenylene diamine	Harmful if swallowed, inhaled or absorbed through skin. Can cause
(OPD):	irritation to eyes, skin mucous membranes and upper respiratory
	tract. Carcinogen
Osmium tetroxide	Osmium tetroxide is extremely hazardous. Very toxic. Flammable.
	Extremely harmful to eyes, respiratory system and skin. Readily
	volatilizes at room temperature. May produce irreversible effects.
	Danger of cumulative effect Always handle in the fume cupboard
	using protective gloves. Dispense over a safety tray to contain any
	spillage. Contaminated glassware and used osmium tetroxide
	fixative should be neutralized in 10% stannous chloride for
	24–48 h, and then rinsed thoroughly with water
Paraformaldehyde	Paraformaldehyde is harmful if inhaled or if allowed to come into
	contact with the skin. Inhalation of the vapour may cause irritation
	to the mucous membranes, upper respiratory tract, eyes and skin.
	Sensitization with allergic, respiratory and skin reactions may
	occur. Handle in the fume cupboard, and wear gloves
Penicillin G	Harmful
Periodic acid	Contact with combustible material may cause fire; causes burns
Phenylmethylsulfonyl	Very toxic by inhalation, in contact with skin and if swallowed;
fluoride (PMSF)	causes burns, contact with water liberates extremely flammable
	gases
Potassium iodide	Harmful if swallowed, inhaled or absorbed through skin. Causes eye
	and skin irritation. Irritable to mucous membranes and upper
	respiratory tract. May cause allergic, respiratory and skin reactions.
Potassium permanganate	Harmful oxidizing agent
Presept	Harmful if swallowed
Propylene oxide	Harmful. Highly flammable. Irritating to eyes, respiratory system and
	skin. May form an explosive vapour mixture. Wear suitable
	protective clothing, gloves and face protection
Pyridoxine-HCl	Avoid contact with skin and eyes
Schiff’s reagent	Causes burns; may cause cancer
Sodium azide	May be fatal if inhaled, swallowed or absorbed through skin. May
	cause eye and skin irritation
Sodium cacodylate	Toxic. Danger of arsenic poisoning. Poisonous by inhalation or
	ingestion. Avoid contact with skin and eyes. Do not breathe dust.
	Wear suitable protective clothing, gloves and face protection when
	preparing solutions and in their use. Danger of cumulative effect
Sodium dodecylsulfate	Harmful
Sodium metabisulfite	May be harmful by inhalation, ingestion and skin absorption. Causes
	eye and skin irritation. Persons with allergies and/or asthma may
	exhibit hypersensitivity to sulfites
Sodium tetrathionate	Irritating to eyes, respiratory system and skin
	(continued overleaf )

386 SELECTED REFERENCE DATA FOR CELL AND MOLECULAR BIOLOGY

Sulfuric acid	Highly corrosive. Avoid contact with skin and clothes. Carry out the
	operation over a sink, and use only small quantities. Wear
	protective gloves
Taab resin	Harmful. Can cause inflammation of the skin and mucous
	membranes. Can irritate respiratory system. Do not swallow or
	inhale. Avoid contact with skin and eyes. Over long exposure can
	produce skin sensitization
Taxol	Highly toxic cytotoxic agent
Tetramethylrhodamine	Irritant to eyes and skin
Thiamine-HCl	Avoid contact with skin and eyes
Tris	Irritating to eyes, respiratory system and skin
Trifluoracetate	Highly poisonous inhibitor
Triton X-100	Toxic; irritating to eyes, respiratory system and skin
Uranyl acetate	Very toxic. Radioactive. Irritating to eyes, respiratory system and
	skin. Do not breathe dust. Wear suitable protective clothing, gloves
	and face protection when preparing solutions and in their use.
	Danger of cumulative effect
Virkon	Irritant
Xylene	Toxic; carcinogen; highly flammable – handle in fume cupboard
ZnSO4.7H2O	Irritating to eyes and skin. Eye contact: irrigate thoroughly with
	water for at least 10 min. If discomfort persists obtain medical
	attention. Inhalation: remove from exposure, rest and keep warm.
	In severe cases obtain medical attention

Centrifugation Data

Calculation of centrifugal force

In centrifugation it is important to differentiate between the speed of centrifugation (RPM) and the centrifugal force (RCF or g) since these are often confused. The centrifugal force generated by a centrifuge can be easily calculated from the equation:

RCF = 11.18 × R × (RPM/1000)2

where R is the distance from the centre of rotation in centimetres, that is the centrifugal force increases as the particles move down the centrifuge tube. As a general rule, the greater the centrifugal force the shorter the separation time. However, centrifugation also generates hydrostatic forces within the solution and so excessive centrifugal forces can disrupt some biological particles such as ribosomes.

			CENTRIFUGATION DATA		387
Applications of centrifuge rotors
Type of rotor		Type of separation
	Pelleting	Rate-zonal	Isopycnic	Isopycnic
			(organelles)	macromolecules
Fixed-angle	Excellent but	Poor	Poor	Excellent
	pellet on side
Swinging bucket	Inefficient but	Excellent	Excellent	Acceptable
	small pellet
Vertical tube	Do not use	Good	Good	Excellent
Zonal rotor	Do not use	Excellent	Excellent	Acceptable

Calculation of pelleting times

Pelleting is the separation of particulate and non-particulate material and it is one of the simplest, most frequently used centrifugation techniques typically as part of a procedure for harvesting cells or the isolation of precipitated material. As a very approximate guide to the conditions needed to sediment various biological particles the following can be used:

Eukaryotic cells	200g for 10 min
Nuclei	1000g for 10 min
Mitochondria	10 000g for 10 min
Microsomes	100 000g for 60 min

As a general rule differential centrifugation produces enriched fractions rather than purified fractions, for example, the ‘nuclear pellet’ obtained by differential centrifugation almost always contains mitochondrial material that has co-sedimented with the nuclei. Similarly, the ‘mitochondrial pellet’ always contains material from lysosomes and peroxisomes. Hence differential centrifugation is usually carried out as an early step in the purification of subcellular components, often prior to further purification involving the use of rate-zonal or, more frequently, isopycnic gradient centrifugation.

The time to pellet particles depends on the k-factor of the rotor; the smaller the k- factor of a rotor the more efficient it is for pelleting particles. The time in hours for pelleting particles of known size (s-value) such as ribosomes can be calculated from the formula:

T = k-factor/s-value

But this formula does assume that sedimentation is taking place in a liquid with the same density and viscosity as water; in sucrose solutions the time required is significantly greater.

Care of centrifugation equipment

One of the major problems when using centrifuges is the corrosion of centrifuge rotors, particularly those made of aluminium alloys. Rotors made of aluminium alloys are very susceptible to severe corrosion even when left to soak in water overnight. Solutions left

388 SELECTED REFERENCE DATA FOR CELL AND MOLECULAR BIOLOGY

in aluminium rotors can cause internal corrosion of the metal alloy. After use always rinse, drain and dry rotors to avoid corrosion and the build-up of contamination. Always follow the manufacturers’ recommendations regarding the care of centrifuge rotors. If at all possible use titanium or carbon composite rotors that are not affected by corrosion by aqueous solutions.

Radioisotope Data

Radioisotope	Half-life	Type of	Shielding	Comments
		radiation	required
3H	12.3 yr	β	None
14C	5760 yr	β	None
22Na	2.6 yr	β	Yes
32P	14.3 days	β	Yes, 10 mm Perspex
33P	25.4 days	β	Yes, 10 mm Perspex	Safer than 32P
35S	87.1 days	β	None	Volatile
51Cr	28 days	EC γ	Yes, lead shielding
59Fe	46.3 days	βγ	Yes, lead shielding
75Se	121 days	EC γ	Yes, lead shielding
125I	60 days	γ	Yes, lead shielding	Volatile
131I	8.1	β, γ	Yes, lead shielding	Volatile

Nuclease inhibitors

Nuclease inhibitors are included in gel electrophoresis procedures to prevent nucleic acid degradation during electrophoresis. Table 7.1 lists the main nuclease inhibitors used in many laboratory procedures. The RNase inhibitor diethyl pyrocarbonate (DEPC) is used in the water and autoclaved used to make up all gel solutions. DEPC-treated water should be autoclaved prior to use to prevent DEPC interfering with any further analyses. Inhibitors are also often used during the isolation of RNA and DNA to prevent degradation by the nucleases present in the host cells.

General manipulation procedures should also be observed to prevent nuclease action:

1.Wear plastic surgical gloves during all manipulations;

2.Autoclave or filter-sterilize all solutions;

3.Wipe surfaces clean prior to starting experiments;

4.Avoid leaning over or breathing into samples;

5.Autoclave or bake at 200 ◦C all utensils prior to experiments.

		RADIOISOTOPE DATA		389
Table 7.1 Nuclease inhibitors and their working concentrations
Agent	Active	Method of nuclease	Comments
	concentration	inactivation
Aurintricarboxylic	10 µM	Complexes to a wide
acid (ATA)		range of nucleases
Bentonite	3 mg/ml	Inactivation by adsorbing
		to nucleases
Diethyl pyrocarbonate	0.1%	Alkylates proteins	Toxic
(DEPC)		disrupting protein
		structure
Dithiothreitol (DTT)	1 mM	Reduces disulfide bonds,
		denaturing proteins
EDTA	1–10 mM	Chelates divalent cations
		needed for ribonuclease
		activity
Guanidine	8 M	Inactivates ribonucleases	Toxic
hydrochloride
Guanidium	4 M	Inactivates ribonucleases	Toxic, strongest
thiocyanate			agent for
			ribonuclease
			inactivation
Heparin	0.5 mg/ml	Binds to basic
		ribonucleases
8-Hydroxyquinoline	0.1% (w/v)	Inactivates ribonucleases	Very toxic
Macaloid	0.015% (w/v)	Adsorbs to ribonucleases
2-Mercaptoethanol	0.1–0.25 M	Reduces disulfide bonds,	Very toxic
		denaturing proteins
Phenol/chloroform	50% (v/v)	Denatures ribonucleases	Toxic
Polyvinyl sulfate	1–10 µg/ml	Complexes to basic
(PVS)		nucleases
Proteinase K	100–200 µg/ml	Hydrolysis of proteins
Sodium dodecyl	0.1–1%	Disrupts protein structures
sulfate
Ribonucleoside	10 mM	Binds to active site of
vanadyl complex		ribonuclease

Index

Page numbers followed by f indicate figures; page numbers followed by t indicate tables.

Aβ1 – 42 peptide preparation of, 349

Abbe´ refractometer, 102 Acetone, 218 Achromat, 5

Acid phosphatase, 94 Acrylamide, 371 Adherins, 52 Agarose, 22

Agarose encapsulation for cell suspensions, 39

Agarose gel, 39

Alcian blue-coated mica, 30 Alcian blue-treated mica, 32 Alkaline phosphatase, 97, 260

non-specific, 141

Alkaline phosphodiesterase, 97 assay for, 143

Alkylation damage, 256 Alzheimer’s disease (AD), 348 Amphibian Ringer’s solution, 242 Amphotericin, 364

Amylases, 96 Amyloid-ß

and enzymes, interactions between, 359, 363

fibrillogenesis, 359 phosphorylation of, 364, 368

phosphorylation by cyclin-dependent kinase-1 (CDK-1), 367, 368

neurotoxicity, 359

peptide, synthesis of 359, 364, 368 phosphorylated

extraction from cells, 366 immunoassay measurement of,

366, 367

Amyloid precursor protein, 345 Amyotrophic lateral sclerosis (ALS), 337 Anode buffer, 182

Anti-apoptotic proteins, 272 Anti-BrdU antibody, 212, 216 Anti-sense amino acid

identification of, 355t Anti-sense peptides, 353, 358

comparison with known proteins, 354, 357 derivation of sequences, 354

direct binding to target proteins, 357, 358 synthesis of 353, 354, 358

Anticoagulant, 80 Antigenic epitopes, 23 Antigenicity, 23, 38, 70

Antineuraminidase antibody, 283 Apical and basolateral domains

fractionation from Caco-2 cells, 163, 164 fractionation from MDCK cells, 165, 166

Apoptosis, 259

immunocytochemical methods to study, 260, 262

inhibition of, 261, 262 Asialoglycoprotein

endocytosis by rat liver, 191, 192, 193 Aspartic acid, 256

Assembly promotion, 329 A-type lamins, 226 Autoradiograms, 229 Autoradiography, 229 Avidin, 23

Axial resolution, 14

Ball-bearing homogenizer, 89, 99, 110, 164, 165, 172, 174, 176, 188, 189

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