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Citometria de fluxo

Faculdade de Medicina da Universidade Federal da Bahia. Citometria de fluxo. Com material obtido em parte da: Purdue University Cytometry Laboratories ( http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm ) Agradecimentos: Dr. Robert Murphy , Carnegie Mellon University, Pittsburgh, PA

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Citometria de fluxo

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  1. Faculdade de Medicina da Universidade Federal da Bahia Citometria de fluxo Com material obtido em parte da: Purdue University Cytometry Laboratories (http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm) Agradecimentos: Dr. Robert Murphy, Carnegie Mellon University, Pittsburgh, PA Dr. J. Paul Robinson, Purdue University Cytometry Laboratories Dr. Carleton Stewart,RPCI Laboratory of Flow Cytometry, Buffalo,NY B. H. Davis, William Beaumont Hospital, Royal Oak, Michigan

  2. O que é a citometria de fluxo:

  3. Avaliação de sub-populações celulares: quadrantes CD4 e CD8. 1 2 45% 2% 100 Log PE Fluorescence (CD4) 10 1 3 4 27% 26% .1 .1 1 10 100 1000 Log FITC Fluorescence (CD8)

  4. DCF Avaliação de ativação celular: explosão respiratória. DCFH-DA DCFH-DA DCFH H O 2 2

  5. G0 G1 G2 M 4N 2N Análise de divisão celular. M G0 G2 DNA Analysis G1 s Count s 0 200 400 600 800 1000 DNA content

  6. Análise de DNA 300 225 150 Counts 75 0 0 200 400 600 800 1000 4N 2N PI Fluorescence

  7. Stimulation 108 0 36 72 144 180 Time (Seconds) Influxo de cálcio

  8. Análise de reticulócitos RMI = 34 RMI = 0 R3 R1 R4 R2 R3 R1 R4 R2 1000 100 .1 1 1000 1 10 10 100 .1 log Thiazole Orange log Thiazole Orange

  9. Citômetro de fluxo Detector & Mechanical Computer System Fluidics

  10. Injector Tip Sheath fluid Fluorescence signals Focused laser beam Célula de Fluxo

  11. Laser FALS Sensor 90LS Sensor 90 Degree Light Scatter

  12. FALS Sensor Fluorescence Fluorescence detector (PMT3, PMT4 etc.) Detectores de fluorescência

  13. Wavelength 400 nm 500 nm 600 nm 700 nm Excitation Emission Fluorescein (FITC) Marcadores fluorescentes

  14. 350 457 488 514 610 632 300 nm 400 nm 500 nm 600 nm 700 nm Cromógenos Common Laser Lines PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid

  15. CD # = cluster designation number Imunofenotipagem: marcação celular CD2 CD4

  16. Imunofenotipagem 100 1000 10 1 .1 Log FITC

  17. Light scatter gating Side Scatter Projection Neutrophils Scale 1000 200 100 50 40 Monocytes 30 20 15 Lymphocytes 8 200 400 600 800 1000 0 90 Degree Scatter

  18. Avaliação de sub-populações celulares: quadrantes CD4 e CD8. 1 2 45% 2% 100 Log PE Fluorescence (CD4) 10 1 3 4 27% 26% .1 .1 1 10 100 1000 Log FITC Fluorescence (CD8)

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