Biofiles Volume 6, Number 5 Centrifugation Centrifugation Basics Density Gradient Media Cell Viability and Proliferation Organelle Isolation Biofilesonline Biofilescontents Your gateway to Biochemicals and Reagents for Life Science Research Introduction 3 Biofi les Online allows you to: Centrifugation Basics 4 • Easily navigate the content of the current Biofi les issue Centrifugation Separations 6 • Access any issue of Biofi les • Subscribe for email notifi cations Histopaque® Troubleshooting Guide 14 of future eBiofi les issues Register today for upcoming issues and Cell Viability and Proliferation 17 eBiofi les announcements at sigma.com/biofi les Organelle Isolation 22 Highlights from this issue: ECACC® Cell Lines 26 Centrifugation is one of the most basic of laboratory applications and is used Centrifugation Equipment 27 by a wide range of clinical and research personnel. However, information on centrifugation theory and separation Cover: The ferris wheel is an amusement techniques are usually only found in park ride making use of centrifugal force centrifuge instrument manuals or by contacting the manufacturers and the Earth's gravitational field. Riders of density gradient media directly. This issue of Biofiles is intended to feel "heaviest" at the bottom of the axis provide a basic understanding of how biological particles behave in of rotation and "lightest" at the top. a centrifugal field and the types of density gradient media used to separate them. Coming Next Issue: The next issue of Biofi les will focus on the challenging problem of cell culture contamination. Cell cultures are vulnerable to a wide variety of contaminants including: • Chemical • Viral • Bacterial, including mycoplasma • Fungal • Insects • Cross contamination with other cell lines This issue will address what Sigma can off er to combat cell culture contamination. Technical content: Mark Frei Technical Marketing Specialist [email protected] Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 3 Introduction Mark Frei Technical Marketing Specialist [email protected] Separation of particles by sedimentation In 1977, the stabilized silica colloid coated is one of the most powerful tools in with polyvinylpyrrolidone (PVP) called biology. Even though sedimentation using Percoll® became available for separating centrifugation is not a new technology, it cells and subcellular particles. In 1968, is essential for cutting edge genomic and Boyum described methods for the isolation proteomic research by providing purified of mononuclear cells from circulating particles of interest. In a survey at the US blood and bone marrow using mixtures of National Institutes of Health, over 65% polysaccharide and a radiopaque contrast of research workers replied that using medium. This led to the development of centrifugation to purify cells, subcellular the first nonionic iodinated density gradient organelles, viruses, proteins, and nucleic medium, metrizamide, in the 1970s.3 Now, acids is an integral part of their work.1 a large selection of commercial iodinated Density gradient centrifugation is a density gradient media are available. technique that allows the separation of This issue of Biofiles examines the theory particles on the basis of their size, shape, and practice of biological separation. The and density. A density gradient is typically beginning section provides a primer on created by layering media of increasing the basic concepts of centrifugation. Three density in a centrifuge tube. When a sample basic types of centrifugal separations are is layered on top of a density gradient and highlighted; differential centrifugation, centrifuged, the various particles move rate-zonal centrifugation, and isopycnic through the gradient at different rates. The centrifugation. A concise description of each particles appear as bands or zones in the is given along with the separation principles gradient with the more dense and larger involved. Cell viability kits, technical support particles migrating furthest. information, and centrifugation equipment A number of different compounds have are also included. been investigated as density gradient We hope you find the reference information media. One of the first density gradient and products relevant. For a comprehensive centrifugation techniques was developed list of our centrifugation media products, in the 1950s and used a buffered sucrose please visit sigma.com/handh solution for the purification of cell organelles. Sucrose quickly became the density medium References: of choice for separating homogenized 1. Biological Centrifugation, J. Graham, p. 1 2. Centrifugation, A Practical Approach (2nd Edition), D. mammalian tissues. Later, cesium chloride Rickwood, p.35 gradients were used to separate DNA of 3. Iodinated Density Gradient Media, A Practical Approach, D. different densities. Meselson and Stahl in Rickwood, p.1 1958 used cesium chloride density gradient centrifugation in an elegant experiment to support the semi-conservative model of DNA replication. Colloidal-silica suspensions were first manufactured by DuPont and sold under the name of LUDOX®.2 4 Centrifugation Basics Centrifugation Basics The earth's gravitational force is sufficient to Most particles are so small that gravitational d2 (p–L) 3 g separate many types of particles over time. v = force is insufficient to overcome the random A tube of anticoagulated whole blood left 18 n molecular forces of particles to influence standing on a bench top will eventually separation. Centrifugation, the name given v = sedimentation rate or velocity of the sphere separate into plasma, red blood cell and white d = diameter of the sphere to separation applications which involve blood cell fractions. However, the length p = particle density spinning around an axis to produce a of time required precludes this manner of L = medium density centrifugal force, is a way to increase the n = viscosity of medium separation for most applications. In practice, g = gravitational force magnitude of the gravitational field. The centrifugal force is necessary to separate most particles in suspension experience a radial particles. In addition, the potential degradation Figure 1. The Stokes equation. centrifugal force moving them away from the of biological compounds during prolonged axis of rotation.2 The radial force generated From the Stokes equation five important storage means faster separation techniques by the spinning rotor is expressed relative to behaviors of particles can be explained: are needed. the earth's gravitational force and therefore is 1. The rate of particle sedimentation is The rate of separation in a suspension of known as the relative centrifugal force (RCF) proportional to the particle size. particles by way of gravitational force mainly or the "g force." The g force acting on particles depends on the particle size and density. 2. The sedimentation rate is proportional is exponential to the speed of rotation Particles of higher density or larger size to the difference in density between the (defined as revolutions per minute; rpm). typically travel at a faster rate and at some particle and the medium. Doubling the speed of rotation increases the centrifugal force by a factor of four. The point will be separated from particles less 3. The sedimentation rate is zero when the centrifugal force also increases with the dense or smaller. This sedimentation of particle density is the same as the medium distance from the axis of rotation. These two particles, including cells, can be explained density. by the Stokes equation, which describes parameters are of considerable significance 4. The sedimentation rate decreases as the the movement of a sphere in a gravitational when selecting the appropriate centrifuge. medium viscosity increases. field.1 The equation calculates the velocity of Table 1 summarizes the applications that can 3 sedimentation utilizing five parameters (see 5. The sedimentation rate increases as the be classified by the relative centrifugal force. Figure 1). gravitational force increases. Parameters Low speed High speed Ultracentrifuge Speed ranges (r.p.m. x 103) 2–6 18–22 35–120 Maximum RCF (x 103) 8 60 700 Pelleting Applications Bacteria - Yes Yes* Animal and plant cells Yes Yes Yes* Nuclei Yes Yes Yes* Precipitates Some Most Yes* Membrane organelles Some Yes Yes Membrane fractions Some Some Yes Ribosomes/polysomes --Yes Macromolecules --Yes Viruses - Most Yes * Can be done but not usually used for this purpose Table 1. Classes of centrifuges and their applications Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 5 RCF is dependent on the speed of rotation Radius Nomogram instructions: (cm) g Rpm 30 5000 in rpm and the distance of the particles from 7000 6000 4500 1. Measure the radius (cm) from the center 25 5000 the center of rotation. When the speed of 4000 4000 of the centrifuge rotor to the end of test 3000 3500 rotation is given in rpm (Q) and the distance 20 19 2000 tube carrier. 18 3000 (r) is expressed in centimeters, RCF can be 17 1500 16 15 1000 2500 2. Obtain the relative centrifugal force calculated by using the formula in Figure 2. 14 800 700 13 600 necessary for the application. 12 500 2000 400 2 11 3. A straight line connecting the value of the Q 10 300 1500 RCF = 11.18 3 r 3 9 200 1400 radius with the relative centrifugal force 1000 8 150 1300 1200 (g) value will enable the speed of the rotor 7 100 Figure 2. Formula for relative centrifugal force (RCF) 80 1100 70 1000 (rpm) to be read off of the right column. 6 60 50 900 A nomogram can also be used to obtain 40 5 800 30 References: the speed of a centrifuge rotor necessary 700 4 20 1. Laboratory Techniques in Biochemistry and Molecular for a desired RCF (see Figure 3). This 15 600 Biology, P.T. Sharpe, p.18 10 quick estimate is useful for low speed 3 500 2. Biological Centrifugation, J. Graham, p. 3 centrifugation applications. However, it is ABC3. Centrifugation, Essential Data, D. Rickwood, p.12 more accurate to use the RCF calculation for speeds in excess of 10,000 rpm.