Flow Cytometry

Services:

• Sample acquisition at the analyzer, either with technical support, or without, after receiving a proper training.
• Cell Sorting with technical support.
• Data analysis support if required.

The aim of the Cytometry Unit is to get ready and focus on the applications of interest of our users.
Most common applications that routinely are run:

Cell Sorting:

• In purification mode (when the most important is to get a high purity of the cell population of interest from the original sample). Up to 4 cell populations at the same time (4way sorting).
• In enrichment mode (when the most important is to get a high recovery of the cell population of interest from the original sample). So that, purity is slightly compromised. Available in 4-way sorting too.
• In single cell deposition (to get just one single cell or any desired number of cells within a single well using any of the standard multiwelll plates).
• In mixed mode (purify and enrich mode run simultaneously even for 2 cell populations at a time).

Analysis:

• Multicolor Immunophenotyping Characterization.
• Viability Assays.
• Cell Cycle and Proliferation Assays.
• Gene Expression (Fluorescent Protein analysis).
• Etc…

The unit is equipped with 3 flow cytometers from Beckman Coulter Company:

•  1x High Speed Cell Sorter Moflo-XDP: 4 lasers (B-R-V-Y), 11 colors (5+2+2+2) o (5+2+4)
• 2x Gallios Analizers: 3 lasers (B-R-V), 10 colors (5+3+2).

Laser abbreviations and Optical Bench configuration:

Lasers:
V: Violet, 405nm;  B: Blue, 488nm;  Y: Yellow, 561nm;  R: Red, 635nm.

Optical Bench Configuration:
10C [2 + 5 + 3] = 10 Colors or PMT detectors in total and [number of them linked to a specific laser line).

Bedford-Guaus S, Kim S, Mulero L, Vaquero JM, Morera C, Adan-Milanès R, Veiga A, Raya A. (2016) Molecular markers of putative spermatogonial stem cells in the domestic cat. Reprod Domest Anim Nov6

Pulecio J, Alejo-Valle O, Capellera-Garcia S, Vitaloni M, Rio P, Mejía-Ramírez E, Caserta I, Bueren JA, Flygare J, Raya A. (2016) Direct Conversion of Fibroblasts to Megakaryocyte Progenitors. Cell Rep 17(3):671-683.

Tiscornia G, Lorenzo Vivas E, Matalonga L, Berniakovich I, Barragán Monasterio M, Eguizábal Argaiz C, Gort L, González F, Mellet CO, García Fernández JM, Ribes A, Veiga A, Izpisúa Belmonte JC. (2013) Neuronopathic Gaucher's Disease: induced pluripotent stem cells for disease modelling and testing chaperone activity of small compounds. Hum Mol Genet 22 (4): 633-45. Epub 2012 Oct 31.

Sander V, Suñe G, Jopling C, Morera C, Izpisua Belmonte JC. (2013) Isolation and in vitro culture of primary cardiomyocytes from adult zebrafish hearts. Nat Protoc 8(4): 800-9.

Marti M, Montserrat N, Pardo C, Mulero L, Miquel-Serra L, Rodrigues AM, Andrés Vaquero J, Kuebler B, Morera C, Barrero MJ, Izpisua Belmonte JC. (2013) Mcad-mediated intercellular interactions activate satellite cell division. J Cell Sci 126 (Pt 22):5116-31. Epub 2013 Sep 17.

Barrero MJ, Sese B, Martí M, Izpisúa Belmonte JC. (2013) Macro histone variants are critical for the differentiation of human pluripotent cells. J Biol Chem 288(22):16110-6. Epub 2013 Apr 17.

Montserrat N, de Oñate L, Garreta E, et al. Generation of feeder-free pig induced pluripotent stem cells without Pou5f1. Cell Transplant. 2012;21(5):815-825

Montserrat N, Ramírez-Bajo MJ, Xia Y, et al. Generation of induced pluripotent stem cells from human renal proximal tubular cells with only two transcription factors, OCT4 and SOX2. J Biol Chem. 2012;287(29):24131-24138.

Giorgetti A, Marchetto MC, Li M, et al. Cord blood-derived neuronal cells by ectopic expression of Sox2 and c-Myc. Proc Natl Acad Sci U S A. 2012;109(31):12556-12561.

Sánchez-Danés A, Consiglio A, Richaud Y, et al. Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells. Hum Gene Ther. 2012;23(1):56-69.

Robles V, Martí M, Izpisua Belmonte JC. Study of pluripotency markers in zebrafish embryos and transient embryonic stem cell cultures. Zebrafish. 2011;8(2):57-63

Eguizabal C, Montserrat N, Vassena R, et al. Complete meiosis from human induced pluripotent stem cells. Stem Cells. 2011;29(8):1186-1195.

Menchón C, Edel MJ, Izpisua Belmonte JC. The cell cycle inhibitor p27Kip¹ controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist. Cell Cycle. 2011;10(9):1435-1447.

Edel MJ, Menchon C, Vaquero JM, Izpisúa Belmonte JC. (2011) A protocol to assess cell cycle and apoptosis in human and mouse pluripotent cells. Cell Commun Signal 9(1): 8.

Edel MJ, Menchon C, Menendez S, Consiglio A, Raya A, Izpisua Belmonte JC. Rem2 GTPase maintains survival of human embryonic stem cells as well as enhancing reprogramming by regulating p53 and cyclin D1. Genes Dev. 2010;24(6):561-573.

Laso FJ, Almeida J, Torres E, Vaquero JM, Marcos M, Orfao A. (2010) Chronic alcohol consumption is associated with an increased cytotoxic profile of circulating lymphocytes that may be related with the development of liver injury. Alcohol Clin Exp Res 34(5): 876-85.

Raya A, Rodríguez-Pizà I, Guenechea G, et al. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature. 2009;460(7251):53-59.

Aasen T, Raya A, Barrero MJ, et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol. 2008;26(11):1276-1284.

Laso FJ, Vaquero JM, Almeida J, Marcos M and Orfao A. (2007) Chronic alcohol consumption is associated with changes in the distribution, immunophenotype, and the inflammatory cytokine secretion profile of circulating dendritic cells. Alcoholism Clinical and Experimental Research 31(5): 846-854.

Laso FJ, Vaquero JM, Almeida J, Marcos M and Orfao A. (2007) Production of inflammatory cytokines by peripheral blood monocytes in chronic alcoholism: Relationship with ethanol intake and liver disease. Cytometry Part B Clinical Cytometry 72(5): 408-15.

Almeida J, Bueno C, Algueró MC, Sánchez ML, Santiago M, Escribano L, Diaz-Agustin B, Vaquero JM, Laso FJ, San Miguel JF, and Orfao A. (2001) Comparative Analysis of the Morphological, Cytochemical, Immunophenotypical, and Functional Characteristics of Normal Human Peripheral Blood Lineage- / CD16+ / HLA-DR+ / CD14-/lo Cells, CD14+ / Monocytes, and CD16- Dendritic Cells. Clinical Immunology 100(3): 325-338.

Bueno C, Almeida J, Algueró MC, Sánchez ML, Vaquero JM, Laso FJ, San Miguel JF, Escribano L, and Orfao A. (2001) Flow Cytometric Analysis of Cytokine Production by Normal Human Peripheral Blood Dendritic Cells and Monocytes: Comparative Analysis of Different Stimuli, Secretion-Blocking Agents and Incubation Periods. Cytometry 46: 33-40.

Bueno C, Almeida J, Algueró MC, Sánchez ML, Cañizo MC, Fernández ME, Vaquero JM, Laso FJ, Escribano L, Díaz-Agustin B, San Miguel JF, y Orfao A. (2000) Caracterización morfológica, histoquímica, fenotípica y funcional de las células dendríticas humanas. Methods Find Exp Clin Pharmacol 22(1): 3-10.

Laso FJ, Vaquero JM, Bueno C, Almeida J, and Orfao A. (2000) Response. Alcoholism: Clinical and Experimental Research 24(2): 241-243.

Almeida J, Bueno C, Algueró MC, Sánchez ML, Cañizo MC, Fernández ME, Vaquero JM, Laso FJ, Escribano L, San Miguel JF, and Orfao A. (1999) Extensive characterization of the imnuphenotype and pattern of citokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage- cells. Clin Exp Immunol 118: 392-401.

Almeida J, Bueno C, Algueró MC, Sánchez ML, Cañizo MC, Fernández ME, Vaquero JM, Laso FJ, Escribano L, San Miguel JF, y Orfao A. (1999) Subpoblaciones de células dendríticas normales: fenotipo y patrón de secreción de citocinas. Haematologica 84(1): 408-414.

Almeida J, Bueno C, Algueró MC, Sánchez ML, Vaquero JM, San Miguel JF, and Orfao A. (1999) Imnunophenotype and citokine production by normal human peripheral blood dendritic cell subsets. Haematologica vol. 84, EHA-4 Abstract Book: 136.