Flow Cytometry

• DESCRIPTION
• ACTIVITIES

• The service permits to: 1) obtain highly purified cell populations from various human and animal tissues by high throughput cell sorting; 2) phenotypically characterize isolated populations and/or any cell type circulating in human or laboratory animal peripheral blood; 3) monitor cell proliferation, survival and differentiation following in vitro and/or in vivo drug treatments. The Flow Cytometry and Cell Sorting service works in collaboration with the Biobank and Microscope services in characterizing cells of interest.

  

  Advanced immunophenotypic analysis of total memory CD45RA- CD8+ T cells.

  

  Evaluation of cell apoptosis using AnnexinV-FITC/PI

• • Studies of ploidy levels using propidium iodide staining and evaluation of cell cycle phases with monoparametric analysis.
  • Cell proliferation: quantitative evaluation of the different cell cycle phases (G0/G1, S, G2/M) by BrdU incorporation and detection by double labelling with anti-BrdU antibody/PI.
  • Quantitative evaluation of cell apoptosis using AnnexinV/PI or 7AAD.

  

  Evaluation of cell cycle using BrdU-FITC/PI

  • Characterization of cell subpopulations circulating in human or laboratory animal peripheral blood: identification of lymphocyte, monocyte and granulocyte subpopulations by physical parameters (forward scatter and side scatter) and their immunophenotypic characterization through mono- or multiparametric analysis.

  

  Immunophenotypic analysis of mouse BAL

  • Monitoring transfection efficiency by green fluorescent protein (GFP) detection, and possibility of sorting GFP-positive cells.
  • Monitoring of cell differentiation through the analysis of lineage-specific surface markers.
  • Phenotypic characterization of specific cell populations, through multiparametric analysis, with the possibility of using up to 9 different fluorochromes, and subsequent physical separation of cells of interest in viable conditions.

  

  Evaluation of cytokines expression in T memory cells

  • Isolation of highly purified viable cell populations (identified by specific surface markers) from different human and animal tissues, using high throughput cell sorting.

  

  Evaluation of mitochondrial activity with MitotrackerRed CMXROs

  These applications concern both the in vitro evaluation of the effects produced by drugs, cytokines or other compounds and also the research aimed at broadening the clinical understanding of various diseases.

  

  CD107a degranulation assay to evaluate immune cell cytotoxic activity

• BD FACSAria IIIu Cell Sorter
• 4-colour BD FACSCalibur Analyzer

• 

  BD FACSAria IIIu Cell sorter

  High performance cytometer and cell sorter, provided with 4 laser sources (blue - 488 nm, red - 633 nm, violet - 405 nm, near UV - 375 nm), it can detect up to 13 parameters, 11 of which can be fluorescence parameters.

  The number of installed lasers, in combination with the revolutionary optical bench, greatly increases the number of fluorescences that the instrument is capable of analyzing at one time and consequently the range of usable fluorochromes.

  The higher performance of the excitation and fluorescence detection system, the electronics and the FACSDiva software 9, together with the easiness of use and the quickness of instrument setup, due to the fixed alignment technology, that also ensures a very high reproducibility, make the FACSAria IIIu the ideal cell sorter for modern biomedical research laboratories.

  BD FACSAria IIIu specifications

  • High-speed sorter with a sensitive, fixed-alignment cuvette flow cell
  • 4 lasers with nearUV (375nm), violet (405nm), blue (488nm) and red (633nm) wavelengths
  • Digital acquisition rates of up to 70,000 events/second
  • Sorting rates of up to 20,000 events/second
  • Multicolor analysis of up to 12 parameters
  • 70μm, 85μm, 100μm and 130μm nozzles available
  • Up to 4-way sorting into 1.5ml or 4ml tubes
  • Single cell sorting in 6-, 24-, 96- and 384- well plates or onto microscopic slides.

  Lasers (exitation)	PMTs	BP Filters (emission)	Fluorochromes (examples)
  *Note: nearUV and Violet lasers cannot be used at the same time.
  Blue 488 nm	A FL1	780/80	PE-Cy7, PE-Vio770, Qdot800
  	B FL2	698/40	PerCP, PerCP-Cy5.5, PerCP-Vio700, PerCP-eFluor710, 7-AAD, PI
  	C FL3	616/23	PE-TexasRed
  	D FL4	585/42	PE
  	E FL5	530/30	FITC, Alexa 488, GFP, Sytox Green, CFSE, DCFDA
  	F SSC	488/10	SSC
  Red 633 nm	A FL6	780/60	APC-H7, APC-Vio770, Live/DEAD Near-IR
  	B FL7	730/45	AF700
  	C FL8	660/20	APC
  Violet 405 nm*	A FL9	780/60	BV750, BV785
  	B FL10	530/30	V500, VioBlue, AmCyan, BV510, Pacific Orange, LIVE/DEAD Aqua
  	C FL11	450/40	V450, VioBlue, Pacific Blue, CellTracker Violet, Zombie violet, AF405
  Near UV 375 nm*	A FL9	780/60	BUV737, BUV805
  	B FL10	530/30	Indo-1 (Blue)
  	C FL11	450/40	Hoechst Blue, DAPI

• 

  BD FACSCalibur flow cytometer

  Bench flow cytometer with two light sources spatially separate: an air-cooled argon laser with an emission at 488 nm and a red diode laser (Visible Red Diode Laser) with an emission at 635 nm. The above described optical configuration allow the analysis of up to 6 parameters, 4 of which can be fluorescence parameters, and a great flexibility in the choice of fluorochromes.

  Specifically designed to support a wide range of applications, the FACScalibur is characterized by an high level of automation (instrument control software and digital fluidics control) to guarantee the higher reproducibility of the results.

  Our facility also makes use of the FlowJo software for data analysis (licence dongles available for signing out for short times). This powerful flow cytometry analysis program is compatible with data acquired from any flow cytometry instruments and capable of executing complex analysis, making the process of analisis of multiparametric acquisitions more efficient, easier and faster.

  Lasers (exitation)	PMTs	BP Filters (emission)	Fluorochromes (examples)
  Blue 488 nm	A FL1	530/30	FITC, Alexa 488, GFP, Sytox Green, CFSE, DCFDA
  	B FL2	585/42	PE
  	C FL3	670 LP	PerCP, PerCP-Cy5.5, PerCP-Vio700, PerCP-eFluor710, 7-AAD, PI
  Red 633 nm	D FL4	661/16	APC

Address

Flow Cytometry and Cell Sorting facility
Area Polo Chimico Biomedico ‘CUBO’ – third floor
Via Fossato di Mortara 70 - 44121 Ferrara
+39 0532 455936/718/849
+39 0532 455718

Technical Director

Prof. Elisabetta Melloni
elisabetta.melloni@unife.it
+39 0532 455936

Contact Person

Dott. Fabio Casciano
fabio.casciano@unife.it
+39 0532 455718

Staff

Dr Fabio Casciano
Dr Elisabetta Melloni

Our staff is qualified to i) receive and, if needed, process/stain the samples, ii) characterize and/or isolate cells of interest, iii) analyze results, and iv) give a quality control of them.

Casciano F, Diani M, Altomare A, Granucci F, Secchiero P, Banfi G, Reali E (2020). CCR4+ Skin-Tropic Phenotype as a Feature of Central Memory CD8+ T Cells in Healthy Subjects and Psoriasis Patients. Front Immunol; 11:529. doi: 10.3389/fimmu.2020.00529. PubMed

Diani M, Casciano F, Marongiu L, Longhi M, Altomare A, Pigatto PD, Secchiero P, Gambari R, Banfi G, Manfredi AA, Altomare G, Granucci F, Reali E (2019). Increased frequency of activated CD8+ T cell effectors in patients with psoriatic arthritis. Sci Rep; 9(1):10870. doi: 10.1038/s41598-019-47310-5. doi: 10.1038/s41598-019-47310-5. PubMed

Chiriaco M, Casciano F, Di Matteo G, Gentner B, Claps A, Di Cesare S, Cotugno N, D'Argenio P, Rossi P, Aiuti A, Finocchi A (2018). Impaired X-CGD T cell compartment is gp91phox-NADPH oxidase independent. Clin Immunol; 193:52-59. doi: 10.1016/j.clim.2018.01.010. PubMed

D'Abundo L, Callegari E, Bresin A, Chillemi A, Elamin BK, Guerriero P, Huang X, Saccenti E, Hussein E, Casciano F, Secchiero P, Zauli G, Calin GA, Russo G, Lee LJ, Croce CM, Marcucci G, Sabbioni S, Malavasi F, Negrini M (2017). Anti-leukemic activity of microRNA-26a in a chronic lymphocytic leukemia mouse model. Oncogene; 36(47):6617-6626. doi: 10.1038/onc.2017.269. PubMed

Secchiero P, Voltan R, Rimondi E, Melloni E, Athanasakis E, Tisato V, Gallo S, Rigolin GM, Zauli G (2017). The γ-secretase inhibitors enhance the anti-leukemic activity of Ibrutinib in B-CLL cells. Oncotarget; 8(35):59235-59245. doi:10.18632/oncotarget.19494. PubMed

Feriotto G, Calza R, Bergamini CM, Griffin M, Wang Z, Beninati S, Ferretti V, Marzola E, Guerrini R, Pagnoni A, Cavazzini A, Casciano F, Mischiati C (2016). Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten. Amino Acids 49(3):551-565. doi: 10.1007/s00726-016-2339-4. PubMed

Voltan R, Rimondi E, Melloni E, Gilli P, Bertolasi V, Casciano F, Rigolin GM, Zauli G, Secchiero P (2016). Metformin combined with sodium dichloroacetate promotes B leukemic cell death by suppressing anti-apoptotic protein Mcl-1. Oncotarget 7(14):18965-77. doi: 10.18632/oncotarget.7879. PubMed

Trapella C, Voltan R, Melloni E, Tisato V, Celeghini C, Bianco S, Fantinati A, Salvadori S, Guerrini R, Secchiero P, Zauli G (2016). Design, Synthesis, and Biological Characterization of Novel Mitochondria Targeted Dichloroacetate-Loaded Compounds with Antileukemic Activity. J Med Chem. 59(1):147-56. doi:10.1021/acs.jmedchem.5b01165. PubMed

Agnoletto C, Melloni E, Casciano F, Rigolin GM, Rimondi E, Celeghini C, Brunelli L, Cuneo A, Secchiero P, Zauli G (2014). Sodium dichloroacetate exhibits anti-leukemic activity in B-chronic lymphocytic leukemia (B-CLL) and synergizes with the p53 activator Nutlin-3. Oncotarget 5:4347-60. doi:10.18632/oncotarget.2018. PubMed

Athanasakis E, Melloni E, Gian Matteo Rigolin GM, Agnoletto C, Voltan R, Vozzi D, Piscianz E, Segat L, Dal Monego S, Cuneo A, Secchiero P, Zauli G (2014). The p53 transcriptional pathway is preserved in ATMmutated and NOTCH1mutated chronic lymphocytic leukemias. Oncotarget; 5(24):12635-45. doi:10.18632/oncotarget.2211. PubMed

• Azienda Ospedaliero-Universitaria di Ferrara
• Centro di Riferimento Oncologico (CRO) di Aviano
• IRCCS Burlo Garofolo di Trieste
• Azienda Ospedaliera di Cremona
• Azienda Ospedaliera di Udine

Some of the services/products offered may be provided in collaboration/integration with other services of the LTTA Laboratory.
Each service includes the supply of images and/or files for scientific publication.
Prices are shown without VAT and are subjected to change according to the cost of consumables (every 12 months).