Gradient centrifugation method

10 ml of leukocyte suspension are diluted with 20 ml sterile 0.9% NaCl and 28 ml layered over 10.5 ml of separation fluid in a sterile 14 X 3 cm tube. Smaller quantitites may be used but the height of the layers should be approximately the same. After centrifugation at 400 g for 25 min at 20°C the red cells are clumped by Ficoll and sediment to the bottom of the tube while the lymphocytes form a 

A more direct method recommended by Pharmacia (Appendix 3) uses 2 ml whole anticoagulant-treated blood diluted with an equal volume of BSS. This is layered over 3 ml Ficoll-Paque and sedimented at 400 g for 30 min. The lymphocytes may be removed from the interphase and the upper layer may be used as a source of autologous plasma. 

Various methods are available for fractionating lymphocytes to obtain pure cultures of the different sorts of white blood cells (Johnstone and Thorpe, 1987). A particularly simple method involves the use of magnetisable polymer particles precoated with monoclonal antibodies (Dynal Ltd. Appendix 3). 

The cells, suspended at 106 per ml of Eagle s MEM supplemented with serine and glycine and 10% autologous serum and containing 50 

of penicillin and streptomycin per ml, are added to 12 X 1.3 cm capped tubes (1 ml), 6 cm Petri dishes (2 ml), 14 X 3 cm tubes (5 ml) or roller bottles (50 ml upwards). The vessels are either loosely capped and incubated in a C02 incubator or are gassed with 5% C02 in air and sealed prior to incubation. 

---

Isolation of Cells for Transport Studies Use of nonequilibrium thermodynamics in the analysis of transport general flow-force relationships and the linear domain, 171, 397 cell isolation techniques use of enzymes and chelators, 171, 444 cell separation by gradient centrifugation methods, 171, 462 cell separation by elutriation major and minor cell types from complex tissues,... 

We wish to report data on a series of whole coals and coal macerals using conventional CP/MAS, dipolar dephasing, and 2-D dipolar dephasing techniques. These data provide a wealth of new structural information and demonstrate that multiple pulse and 2-D spectroscopic techniques can be utilized on complex carbonaceous materials. We also report data obtained on maceral samples separated by the density gradient centrifugation method which separates coal maceral groups according to density. 

Two different methods are in general use the glass bead column method of Rabinowitz (1964, 1973) and the density gradient centrifugation method of Boyum (1968). 

Pinter, G. G., and D. B. Zilversmit A gradient centrifugation method for the determination of particle size distribution of chylomicrons and of fat droplets in artificial fat emulsions. Biochim. biophys. Acta (Amst.) 59, 116—127 (1962). 

Schmitz, J, Petrasch, S, van Lunzen, J, Racz, P, Kleine, H D., Hufert, F, Kern, P., Schmitz, H., and Tenner-Racz, K. (1993) Optimizing follicular dendritic cell isolation by discontinuous gradient centrifugation and use of the magnetic cel sorter (MACS). J Immunol Methods 159, 189-196... 

Moret et al. (67,68) studied all the parameters that influence amine recovery under conditions where a liquid-liquid purification step with an organic solvent follows the acid extraction, prior to derivatization with DBS and RP-HPLC analysis. The optimized methods of sample preparation for different foods, including cheese, meat, and fish, are given. The same research group (69) optimized the extraction conditions for Phe, Put, Cad, His, Tyr, Spe, and Spd. Food samples were first mixed with TCA and centrifuged and then basified and extracted with BuOH/CHCl3 (1 1). The BAs were then derivatized with DNS and separated on a Spherisorb 3S TG column with an ACN-H20 gradient. The method was applied to samples of tuna, salmon, and salami. 

This standard method of density gradient centrifugation, however, has a serious disadvantage. Because of the sedimentation and diffusion equilibrium formation of the density profile the time needed for orie experiment amounts to more than 15 hours. 

For the investigation of the chemical composition of dispersed particles, e. g. in polymer latices, a rapid method of density gradient centrifugation is developed. The method yields in a few minutes the density distribution and by this the distribution of the chemical composition of the particles. The standard density gradient centrifugation needs for the same results because of the time-consuming equilibrium gradient formation more than 15 hours. 

Watanabe, Y., Yamanaka, N., Fukui, Y., (1998), Control of composition gradient in a metal-ceramic functionally graded material manufactured by the centrifugal method , Composites Part A, 29 A, 595-601. 

---