Flow Cytometry Laboratory core facility

In the Instiute of Biochemistry, we have a longstanding experience in the use of flow cytometry (FACS) technology that was implemented in our institution starting 2006.

In the Instiute of Biochemistry, we have a longstanding experience in the use of flow cytometry (FACS) technology that was implemented in our institution starting 2006, with the acquisition of the BD FACSCalibur 1-laser instrument. The PROCERA project allowed the expansion of our technical capacity with the investment in two more machines: the BD FACSVerse 3-laser analyser and the BD FACSAria III 5-laser cell sorter.
Our expertise and instruments enable us to perform most flow cytometric assays that have been described in the literature. These range from routine cell cycle and proliferation analyses to immunophenotyping and multicoulour applications. Also, separation of subpopulations of cells is possible using our cell sorter or Miltenyi MACS magnetic-beads separation system. The technological workflow consists of staining, data collection, quality control, data analysis/interpretation, and creation of graphics.

Available applications are:

  • Sample preparation and staining,
  • Characterization of cell populations based on scattered light intensity measurements, and autofluorescence,
  • Viability assays,
  • Cell cycle measurement,
  • Apoptosis assay (Annexin V/7AAD double staining),
  • Nanoparticle characterization,
  • Cell proliferation studies (CFSE tracking),
  • Function assays (in vitro cell stimulation),
  • Signal transduction pathway analyses (detection of intracellular phosphorylated proteins),
  • Multicolour flow cytometry (two-three color analyses are routinely performed, greater than four color are under development) including characterization of plasma membrane, intracellular, and intranuclear antigens,
  • Cell sorting including viable, sterile, four-way or single-cell sorting

Instruments

FACSCalibur

1-laser (488 nm)

3 colours

Cell QuestPro software analyser

FACSVerse analyser

3-lasers (405, 488, 632 nm)

8 colours

automated laser alignment

Volumetric Flow Sensor  - cell absolut counting standard and high sensitivity modes

Universal loader  - automatic acquisition from tube racks or plates

BD FACSuite software

FACSAriaIII cell sorter

5-lasers (355/405, 488, 561, 633 nm)

up to 18 colours

gel-coupled cuvette flow cell

70, 85, and 100 microns nozzles

up to 70000 events per second

four-way and single-cell sorting capabilities

automated cell deposition unit (ACDU) for slide and

plate sorting: 6-, 24-, 48-, 96- and 384-well plates

BD FACSDiva software

If you are a first time user or are reserving time on the FACSCalibur, FACSVerse or FACSAriaIII (for sorting or multi-color analysis), contact Dr. Livia Sima (021-2239069, room 322) with the appointment request.  If you are a trained user, please feel free to use the scheduler available for each instrument.

To schedule a session of assisted acquisition and/or analysis, a minimum one week advance notice is required.

Links:

Experimental design

SpectraViewer: http://www.bdbiosciences.com/us/s/spectrumviewer

https://www.biolegend.com/spectraanalyzer

https://www.chroma.com/spectra-viewer

https://www.thermofisher.com/order/spectra-viewer

Fluorish: https://sbmriflowcytometry.wordpress.com/2012/08/10/fluorish-a-tool-for-creating-facs-antibody-panels/

 

Tutorials

Molecular Probes - Introduction to flow cytometry

BD Training and e-Learning

Abcam - Introduction to flow cytometry

 

Societies:

ISAC

Romanian Association for Flow Cytometry

 

Online resources (blogs):

EuroFlow

U of C Flow cytometry Blog

Purdue Cytometry Archive Search

Current Protocols In Cytometry

 

Journals:

Cytometry Part A

Cytometry Part B

 

Software:

Cytobank

FlowJo (Tree Star, Inc.)

FCS Express (De Novo Software)

WinMDI

Modfit (Verity Software House) for cell cycle distribution analysis

 

Main vendors of FACS reagents and kits:

  • Becton Dickinson
  • BioLegend
  • eBiosciences
  • Thermo Scientific

Recommended literature:

Interpreting flow cytometry data: a guide for the perplexed

Standardization of Cytokine Flow Cytometry Assays

Guidelines for the Presentation of Flow Cytometric Data

Intracellular Phospho-Protien Staining Techniqes for Flow cytometry: Monitoring Single Cell Signaling Events

 

A list of our publications:

  • Ionescu AE, Mentel M, Munteanu CVA, Sima LE, Martin EC, Necula-Petrareanu G, Szedlacsek SE. "Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation", International Journal of Molecular Sciences, (2019)
  • Mihaela Uta, Livia Elena Sima, Patrik Hoffmann, Valentina Dinca, Norica Branza-Nichita, Development of a DsRed-expressing HepaRG cell line for real-time monitoring of hepatocyte-like cell differentiation by fluorescence imaging, with application in screening of novel geometric microstructured cell growth substrates, Biomedical microdevices 19 (1), 3 (2017)
  • Corotchi MC, Popa MA, Remes A, Sima LE, Gussi I, Plesu ML. "Isolation method and xeno-free culture conditions influence multipotent differentiation capacity of human Wharton.s jelly-derived mesenchymal stem cells., Stem Cell Res.Ther., 4, 81 (2013)
  • Dinca V, Florian PE, Sima LE, Rusen L, Constantinescu C, Evans RE, Dinescu M, Roseanu A "MAPLE-based method to obtain biodegradable hybrid polymeric thin films with embedded antitumoral agents.., Biomed.Microdev., DOI 10.1007/s10544-013-9801-9, July (2013)
  • Ivan V, Martinez-Sanchez E, Sima LE, Ooschot V, Klumperman J, Petrescu SM, Van der Sluj P."AP-3 and Rabip4' Coordinately Regulate Spatial Distribution of Lysosomes.", PLoS One., 7(10), e48142 (2012)
  • Filimon A, Sima LE, Appelhans D, Voit B, Negroiu G."Internalization and Intracellular Trafficking of Poly(propylene imine) Glycodendrimers with Maltose Shell in Melanoma Cells.", Curr.Med.Chem., 19(29), 4955-4968 (2012)
  • Florian P, Macovei A, Sima LE, Nichita N, Mattsby-Baltzer I, Roseanu A."Endocytosis and trafficking of human lactoferrin in macrophage-like human THP-1 cells.", Biochem Cell Biol., 90(3), 449-455 (2012)
  • Sima LE, Stan GE, Morosanu CO, Melinescu A, Ianculescu A, Melinte R, Neamtu J, Petrescu SM "Differentiation of mesenchymal stem cells onto highly adherent radio frequency-sputtered carbonated hydroxylapatite thin films.", J Biomed Mater Res A, 95(4), 1203-1214 (2010)
  • Roseanu A, Florian PE, Moisei M, Sima LE, Evans RW, Trif M "Liposomalization of lactoferrin enhanced its anti-tumoral effects on melanoma cells.", Biometals, 23(3), 485-492 (2010)
  • Negroiu G, Piticescu RM, Chitanu GC, Mihailescu IN, Zdrentu L, Miroiu M "Biocompatibility evaluation of a novel hydroxyapatite-polymer coating for medical implants (in vitro tests)",J.Mat.Sci:Mat.Med, 19, 1537-1544 (2008)