ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 359 (2006) 283–284 www.elsevier.com/locate/yabio
Notes & Tips
A homemade cytospin apparatus Giorgia Sisino, Daniele Bellavia, Maria Corallo, Fabiana Geraci, Rainer Barbieri ¤ Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Palermo, 90128 Palermo, Italy Received 14 July 2006 Available online 26 September 2006
The cytospin procedure uses low-centrifugal force to separate and deposit a monolayer of cells on slides, maintaining cellular integrity. A purpose-built centrifuge or
rotor is needed to obtain thin-layer cell preparations in a clearly deWned round or rectangular area on the slide (for details and Wgures see [1]). We developed a simple inexpensive system using disposable pipette tips and a common desk centrifuge which provides results identical to those for conventional cytospin devices. In the system that we developed, a piece of 3MM Wlter paper (Whatman Int. Ltd., Maidstone, England) is inserted into a 0.1- to 10.0-l micropipette tip and the top is cut as shown in Fig. 1A. A second uncut tip is inserted into the Wrst and the two-tip system is placed on a pretreated glass slide (Bio Optica, Milan, Italy) (Fig. 1C). The second (upper) tip is driven 2 or 3 mm from the slide. A plastic slide, of dimensions not exceeding the glass slide and 3MM paper dimensions, previously pierced to produce holes the diameter of which exceeds the diameter of the tip by about 1 mm (Fig. 1B) is used. This slide is placed around the tips, holding them Wrm on the glass slide and squeezing the paper which there acts as a washer, as shown in Figs. 1C and 1D. After positioning
Fig. 1. Schematic drawing of the assembly of the system. *
Corresponding author. Fax: +39 09 16577210. E-mail address:
[email protected] (R. Barbieri).
0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.09.002
Fig. 2. Positioning of the assembled system in a common desk centrifuge.
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Fig. 3. Embryonic mouse Wbroblasts (4 £ 105) spotted using a conventional cytospin centrifuge (A and C) and using our system (B and D).
and compressing the plastic slide, 3MM Wlter paper surrounds and tightly adheres to the borders of the lower tip (Fig. 1D). The system is then blocked with adhesive tape, as shown in Fig. 2. The sample is loaded in the top of the uncut tip (Fig. 1D), which contains a volume up to 100 l, and the system is placed in a common swing-out desk centrifuge bucket, as shown in Fig. 2. If necessary, glass slides can be cut according to the internal diameter of the centrifuge bucket by using a diamond glass cutter. Centrifugation of the sample is performed at 900g for 5 min. During centrifugation, the stability of the vertical position of the tips with respect to the glass slide surface is maintained by centrifugal force, which acts on all the components of the system. Under centrifugation the cellcontaining sample is forced to pass throughout the upper tip and is distributed in the round area on the slide surrounded by the lower tip border. The liquid phase passes between the lower tip border and the slide and is absorbed by the 3MM Wlter paper, while cells are spotted over the slide. After disassembling the system, cells were dyed with 0.05% toluidine blue (Sigma Aldrich Co.) in phosphatebuVered saline [2]. Fig. 3 shows a suspension of 4 £ 105/ml embryonic mouse Wbroblasts [3] spotted using a conventional cytospin centrifuge (Shandon Southern Cytospin) (A, C) and using our system (B, D). No diVerence in the spreading of cells is visible using the two systems. Cells appear to be monolayered and their morphological integrity is preserved using both procedures.
This system is simple, rapid, inexpensive, and reliable and presents further advantages with respect to conventional cytospin: (1) from one to at least four diVerent samples can be loaded in a single slide using our system (see Fig. 1E; and also Fig. 2, in which a two-tips system is shown), while conventional cytospin allows the loading of a single sample in each slide, (2) no washing procedures of the apparatus are needed because disposable tips are used in the process, and (3) after centrifugation and disassembly of the system, spot borders appear perfectly circular (not shown), indicating that no smearing of the sample occurs during centrifugation procedures. Acknowledgments This work was supported by funds of Italian Ministry of University and Research (60%). We thank Dr. Giuseppina Turturici for her precious suggestions. References [1] www.biomedicalpolymers.com/pages/cyt_funnels.html. [2] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. [3] M.G. Minasi, M. Riminucci, L. De Angelis, U. Borello, B. Berarducci, A. Innocenzi, A. Caprioli, D. Sirabella, M. Baiocchi, R. De Maria, R. Boratto, T. JaVredo, V. Broccoli, P. Bianco, G. Cossu, The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and diVerentiates into most mesodermal tissues, Development 129 (2002) 2773–2783.
