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Plug Flow Cytometry

Bruce S. Edwards¹, Larry A. Sklar^2,1

¹University of New Mexico, Albuquerque, New Mexico
²Los Alamos National Laboratory, National Flow Cytometry Resource, Los Alamos, New Mexico

Publication Name:

Current Protocols in Cytometry

Unit Number:

Unit 1.17

DOI:

10.1002/0471142956.cy0117s17

Online Posting Date:

August, 2001

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Abstract

Although flow cytometry has the powerful ability to rapidly screen large collections of cells, the technology has yet to be efficiently applied to large-scale screening operations involving multiple discrete suspensions. High-throughput flow cytometry would be beneficial to many areas of biological investigation, such as modern drug discovery, which involves testing of cellular targets against millions of potentially valuable compounds. The authors have developed a flow injection analysis approach to automated sample handling in which individual sample suspensions are sequentially inserted as plugs of precisely defined volumes into a flowing fluid which delivers them to the laser beam. The unit describes the basic elements and concepts of this plug flow system and discusses representative applications.

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Unit Introduction
Bibliography
Figures

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Figures

Figure 1.17.1

A schematic of the interposition of sample loops in the two fluidic pathways of the reciprocating valve. A two-position eight-port valve (e.g., VICI C22Z, Valco Instruments) reciprocates between two positions. The entry and exit ports for each of the two fluid pathways that pass through the valve (path 1 and path 2) remain constant. (A) In valve position 1, fluid in path 1 passes through sample loop A while fluid in path 2 passes through sample loop B. (B) In valve position 2, the sample loops are switched so that each is in the alternate fluid pathway. In this fashion, a plug of fluid from each pathway is inserted into the alternate pathway. (C-F) Hypothetical sequence of events illustrating sequential sample plug formation and delivery to the flow cytometer. (C) A bolus of sample particles (gray band) has been moved from the sample source and through the valve so that a 5-µl fraction of sample is contained in one of the valve sample loops. (D) The valve has switched to its alternate position and the 5-µl sample plug has been partially moved out of the sample loop towards the flow cytometer. A second bolus of sample particles (black band) moves into the second sample loop as extraneous particles from the first sample move to waste. (E) The second sample is positioned for plug formation as the first sample plug is delivered to the flow cytometer. (F) After another valve switch, a third bolus of sample particles (gray band) moves into the first sample loop as a 5-µl plug from the second sample moves from the second sample loop to the flow cytometer.

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Figure 1.17.2

System schematics for sample acquisition and delivery of sample plugs to the flow cytometer. (A) Samples are aspirated under the control of a Cavro XL3000 modular digital pump equipped with a 500-µl syringe barrel and attached to the sample-uptake fluid pathway. In the pressurized fluid transport pathway is a 25 × 100–mm Falcon polystyrene tube containing a fluid buffer solution (e.g., phosphate-buffered saline). The tube is sealed at the top with a standard sample cap assembly for the Elite flow cytometer (Beckman Coulter) and pressurized by attaching the silicone tubing part of the cap assembly to the cytometer sample chamber pressure outlet. A low-pressure union (Upchurch Scientific) couples the sample insertion rod of the cap assembly to a length of Teflon tubing that is joined in turn to an inlet port of the reciprocating valve. Inserted into the valve outlet port of the fluid transport pathway is another sample insertion rod. This is joined to the flow nozzle insertion rod by a 1-cm length of silicone tubing (sample tubing specified for conventional use with the Elite). TT, Teflon tubing of 0.065-in. o.d. and 0.01-in. i.d. (Upchurch Scientific). Each sample loop is a 10-cm length of this tubing and holds a fluid volume of 5 µl. (B) Use of a peristaltic pump in the sample uptake fluid pathway. A Gilson Minipulse 3 peristaltic pump (Gilson, Inc.) is used with 0.02-in. i.d. Gilson peristaltic tubing to aspirate and deliver samples to the reciprocating valve. Other system components are the same as in panel A.

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Figure 1.17.3

Light-scatter gating to enable fluorescence analysis of the best optically aligned particles. Flow Check beads (Beckman Coulter) were analyzed at a volumetric sample-stream flow rate of 0.8 µl/sec to determine the light-scatter profile (forward scatter versus log side scatter) (A) and the green fluorescence intensity profile (B) of optimally aligned beads. The beads were then repetitively sampled by plug flow cytometry at a fluid transport stream flow rate of 3.6 µl/sec to determine the light scatter (C) and fluorescence intensity (D) profiles. The electronic light-scatter gate enclosing optimally aligned beads in panel A was used to gate the fluorescence intensity analysis in D (filled histogram) for comparison with the fluorescence profile of ungated beads (open histogram).

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Figure 1.17.4

Positioning of the reciprocating valve above the flow nozzle. The valve is attached to an articulating-arm mounting system (Edmund Scientific), which is mounted on a 12 × 12–in. optical bench plate (Edmund Scientific). The plate rests atop the flow cytometer in proximity to the flow nozzle. A linear translation stage (Edmund Scientific) between the arm and the valve is used for fine positioning.

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Figure 1.17.5

Repetitive sampling to characterize the precision of particle concentration determinations. A stirred suspension of Flow Check beads was sampled at 3.5 (A), 4.5 (B), and 7 (C) sample plugs/min. The mean ±SD number of beads in sample plug peaks (bottom of each panel) were 502 ± 43, 500 ± 35, and 847 ± 42, respectively. This indicated source concentrations of 1.0 × 10⁵, 1.0 × 10⁵, and 1.7 × 10⁵ beads/ml in the respective sample suspensions. Dot clusters at the top of each panel represent the log fluorescence intensity profile of individual beads on the time axis.

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Literature Cited

Literature Cited
	Edwards, B.S., Kuckuck, F., and Sklar, L.A. 1999. Plug flow cytometry: An automated coupling device for rapid sequential flow cytometric sample analysis. Cytometry 37:156-159.
	Edwards, B.S., Curry, M.S., Tsuji, H., Larson, R.S., Brown, D., and Sklar, L.A. 2000. Expression of P-selectin at low site density promotes selective recruitment of eosinophils over neutrophils. J. Immunol. 165:404-410.
	Edwards, B.S., Kuckuck, F.W., Prossnitz, E.R., Ransom, J.T., and Sklar, L.A. 2001a. HTPS flow cytometry: A novel platform for automated high throughput drug discovery and characterization. J. Biomol. Screening 6:83-90.
	Edwards, B.S., Kuckuck, F.W., Prossnitz, E.R., Okun, A., Ransom, J.T., and Sklar, L.A. 2001b. Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology. Cytometry 43:211-216.
	Kachel, V., Fellner-Feldegg, H., and Menke, E. 1990. Hydrodynamic properties of flow cytometry instruments. In Flow Cytometry and Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 27-45. Wiley-Liss, New York.
	Lindberg, W., Ruzicka, J., and Christian, G.D. 1993. Flow injection flow cytometry: A new approach for sample and solution handling in flow cytometry. Cytometry 14:230-236.
	Nolan, J.P. and Sklar, L.A. 1998. The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions. Nature Biotechnology 16:633-638.
	Nolan, J.P., Lauer, S., Prossnitz, E.R., and Sklar, L.A. 1999. Flow cytometry: A versatile tool for all phases of drug discovery. Drug Discov. Today 4:173-180.
	Seamer, L.C., Kuckuck, F., and Sklar, L.A. 1999. Sheath fluid control to permit stable flow in rapid mix flow cytometry. Cytometry 35:75-79.
	Sklar, L.A., Seamer, L.C., Kuckuck, F., Posner, R., Prossnitz, E., Edwards, B., and Nolan, J.P. 1998. Sample handling for kinetics and molecular assembly in flow cytometry. S.P.I.E. Proc. 3256:144-153.
	Zhao, R., Natarajan, A., and Srienc, F. 1999. A flow injection flow cytometry system for on-line monitoring of bioreactors. Biotechnol. Bioeng. 62:609-617.
Internet Resources
	http://www.beyondlogic.org/serial/serial.htm
Interfacing the serial/RS232 port. A very informative series of articles by Craig Peacock relevant to the type of serial port programming used in the Plug Flow system.