ZMBP Shared Resource Lab
Fluorescence Activated Cell Sorting (FACS)
Welcome to the facility for Flow Cytometry and Fluorescence Activated Cell Sorting (FACS) at the Center for Plant Molecular Biology (Zentrum für die Mokularbiologie der Pflanzen) at the University of Tübingen. Flow cytometry is a method for analysing particles in a stream of liquid and in particular, our applications are primarily based on the analysis of fluorescence. Light (photons) can be absorbed by certain molecules leading to excitation of an electron from its ground state to an excited state (an electron orbit of higher energy). In certain instances, this absorbed energy is reemitted as light (photons) of a longer wavelength as the electron returns to the ground state; this is called fluorescence. As long as one is able to analyse their samples in solution, elicit fluorescence and maintain a particle much smaller than that of the nozzle size, one can use flow cytometric methods for applicable analyses. In addition to the measurement of fluorescence, one can get a rough estimate of particle size and particle granularity or surface differences. These attributes are independent of fluorescence. Common applications in plant science have been the measurement of DNA content for determining the phase of the cell cycle, measuring DNA content calculating the ploidy level and counting chromosomes by using fluorescent dyes that are specific to DNA. Another application has been the use of cell line specific GFP markers for the isolation of specific cell populations (e.g. quiescent root cells) by FACS followed by analysis of RNA or potentially protein content. Protein-protein interactions can also be effectively analysed by fluorescence using BiFC techniques when combined with flow. Since no microscope function associated with this particular method (non-imaging cytometry), the determination of sub-cellular compartmentalization must be made independently of the FACS treatment, either before or afterwards. Our MoFlo XDP is capable of currently detecting fluorescence excited by blue laser (488 nm), a yellow-green laser (561 nm) and either a violet laser (405 nm) or UV-laser (375 nm). Our CytoFLEX is equipped with a single 488 nm laser and 4 detection channels. We predominately work with plant material.
MoFlo at ZMBP
Our FACS machine is a MoFlo (Modular Flow) XDP model from Beckman Coulter, giving us a needed flexibility required for working with plant cells. We possess 70uM, 100uM, 150uM (and 200uM) nozzles. All of these nozzles have been successfully tested with our current set-up. Our present configuration uses a 30 - 150mW 488nm Argon laser (blue light), a 1 - 100mW 561 nm green laser for excitation, and a UV 375nm (1 to 50mW) or violet 405nm (1 to 100mW) laser.
The fluorescence is detected through a series of dichromatic mirrors and filters which give us crude spectral data (see insert at the right). In principle, we have filters for green, yellow, orange, red, infra-red light. Thus, with our current filter/mirror set we can detect and easily distinguish GFP/YFP and RFP/mCherry fluorescence. We are open to special applications or other filter arrangements depending on the project and demands as we have a fully modular system.
Sorting (FACS) and/or multicolor Analysis
The MoFlo XDP (BC) allows sorting of up to 4 decisions during a run, with the option to collect abort events as well. For single cell sorting events, the machine is capable of sorting into 96, 384 and 1596 (optimal alignment) well plates. Furthermore, it is possible to create user defined sorting patterns on slides, into plates, tubes, etc.. Use of the XDP requires a principle operator at all times; inquire about training for long-term projects.
Our facility has a CytoFLEX (BC) with a single 488 nm laser and 4 band passes. This is ideal for GFP/YFP, but mRFP and mCherry can also be detected. Chlorophyll fluorescence is also resolvable. After training, you are allowed to use the CytoFLEX at any time. Trouble shooting and problems should be reported to a principle Operator as soon as possible.
The XDP Sample Station is capable of accommodating 0.5 ml and 1.5 ml Eppdendorf tubes, 14 ml, 15 ml round bottom falcon tubes, 15 ml and 50 ml conical bottom falcon tubes. 2 ml Eppendorf tubes are not accepted. 24, 96, 384 well plates and user defined regions.
The CytoFLEX accommodates 5 mL culture tubes, 2.0 and 1.5 ml Eppendorf tubes.
Biological Safety Level
Our facility is strictly S1 (BL1). No exceptions are allowed.
Note: Typically, as most mammalian cell lines carry viral particles or are generated with retroviruses, even if they are S1 (BL1), we have to consider them S2 (BL2). You can still contact us though and we will have a look; make sure you attach the appropriate data sheet(s).
Usage / Services
The MoFlo XDP and CytoFLEX is currently available to all members of the University of Tübingen, with priority given to those who are members of the ZMBP. IF you wish to use the MoFlo, please contact me below. We are running an open schedule with a first-come-first-serve policy, advance bookings can be arranged for special projects (please use the email address listed below). Please be aware that extensive costs will have to be reimbursed in full, but that we currently run on a cost-recovery basis only. Also please read the section above about our Biological Safety Level policy.
We currently use Summit 5.4 software for acquisition and Summit 5.4 for the XDP. You may of course take and process your data yourself with your software of choice. The CytoFLEX comes with its own software: CytExpert. FlowJo can also be provided when doing analysis "in house".
Specific questions about sheath fluids should be personally directed to the principle operator.
Your data is your intellectual property. We will keep archives of the cytometry files and the set-up parameters for at least 10 years. As a user though, you are required to keep track of your samples. We will assist as much as possible if desired though. Therefore, no data or outcomes will be shared with anyone at any time without Your permission.
Flow requests, appointments, questions
facs (a) zmbp.uni-tuebingen.de
Core Manager/contact information
ZMBP - Central Facilities
Kenneth W. Berendzen, Dr. rer. nat.*
Auf der Morgenstelle 32
D-72076, Tübingen, Germany
Office: Tel. +49-(0)7071-29-78721
Lab: Tel. +49-(0)7071-29-78715
Only individuals authorized to use the MoFlo Legacy are allowed to do so without constant supervision from the Principle Operator. *persons who should be addressed first. I will provide training to long term projects for in-house personnel. In this way they can run their applications themselves, but don't worry, continued support and supervision will be provided. Operator training is possible for long term projects.
Current Principle Operators:
(publication) Durka, W., Michalski, S. G., Berendzen, K. W., Bossdorf, O., Bucharova, A., Hermann, J.-M., Hölzel, N. and Kollmann, J. (2016), Genetic differentiation within multiple common grassland plants supports seed transfer zones for ecological restoration. J Appl Ecol. doi:10.1111/1365-2664.12636
(publication) Conserved and unique features of the homeologous maize Aux/IAA proteins ROOTLESS WITH UNDETECTABLE MERISTEM 1 and RUM1-like 1. Zhang Y, Marcon C, Tai H, von Behrens I, Ludwig Y, Hey S, Berendzen KW, Hochholdinger F. J Exp Bot. 2016 Feb;67(4):1137-47. doi: 10.1093/jxb/erv519. Epub 2015 Dec 15.
(publication) The Elucidation of the Interactome of 16 Arabidopsis bZIP Factors Reveals Three Independent Functional Networks. Llorca CM, Berendzen KW, Malik WA, Mahn S, Piepho HP, Zentgraf U. PLoS One. 2015 Oct 9;10(10):e0139884. doi: 10.1371/journal.pone.0139884. eCollection 2015.
(publication) Cooperative action of the paralogous maize lateral organ boundaries (LOB) domain proteins RTCS and RTCL in shoot-borne root formation. Xu C, Tai H, Saleem M, Ludwig Y, Majer C, Berendzen KW, Nagel KA, Wojciechowski T, Meeley RB, Taramino G, Hochholdinger F. New Phytol. 2015 Apr 22. doi: 10.1111/nph.13420
(publication) Cell type-specific transcriptome analysis in the early Arabidopsis thaliana embryo. Slane D, Kong J, Berendzen KW, Kilian J, Henschen A, Kolb M, Schmid M, Harter K, Mayer U, De Smet I, Bayer M, Jürgens G. Development. 2014 Nov 19. pii: dev.116459.
(publication) Diversity of stability, localization, interaction and control of downstream gene activity in the Maize Aux/IAA protein family. Ludwig Y, Berendzen KW, Xu C, Piepho HP, Hochholdinger F. PLoS One. 2014 Sep 9;9(9):e107346.
(publication) Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry. Berendzen KW, Böhmer M, Wallmeroth N, Peter S, Vesić M, Zhou Y, Tiesler FK, Schleifenbaum F, Harter K. Plant Methods. 2012 Jul 12;8(1):25.
(publication) Molecular interactions of ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS, a LOB domain protein regulating shoot-borne root initiation in maize (Zea mays L.). Majer C, Xu C, Berendzen KW, Hochholdinger F. Philos Trans R Soc Lond B Biol Sci. 2012 Jun 5;367(1595):1542-51
(acknowledgement) Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Scheuring D, Viotti C, Krüger F, Künzl F, Sturm S, Bubeck J, Hillmer S, Frigerio L, Robinson DG, Pimpl P, Schumacher K. Plant Cell. 2011 Sep;23(9):3463-81. doi: 10.1105/tpc.111.086918. Epub 2011 Sep 20.
(publication) Rootless with undetectable meristem 1 encodes a monocot-specific AUX/IAA protein that controls embryonic seminal and post-embryonic lateral root initiation in maize. von Behrens I, Komatsu M, Zhang Y, Berendzen KW, Niu X, Sakai H, Taramino G, Hochholdinger F. Plant J. 2011 Apr;66(2):341-53. doi: 10.1111/j.1365-313X.2011.04495.x. Epub 2011 Mar 1.
(publication) Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus. Wanke D, Hohenstatt ML, Dynowski M, Bloss U, Hecker A, Elgass K, Hummel S, Hahn A, Caesar K, Schleifenbaum F, Harter K, Berendzen KW. PLoS One. 2011 Feb 10;6(2):e16070.
(publication) Promoter specificity and interactions between early and late Arabidopsis heat shock factors. Li M, Berendzen KW, Schöffl. Plant molecular biology. 2010 May 11.
(publication) Detection of in vivo interactions between Arabidopsis class A-HSFs, using a novel BiFC fragment, and identification of novel class B-HSF interacting proteins, Li M, Doll J, Weckermann K, Oecking C, Berendzen KW, Schöffl F., Eur J Cell Biol. 2010 Feb-Mar;89(2-3):126-32. Epub 2009 Nov 27.
(acknowledgment) Regulators of PP2C phosphatase activity function as abscisic acid sensors. Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E. Science.2009 May 22;324(5930):1064-8. Epub 2009 Apr 30.
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