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Hematopoietic Stem/Progenitor Cells CFC Assays & Immunophenotype

Explore the regulation, heterogeneity, and cell surface markers of hematopoietic stem/progenitor cells through CFC assays and immunophenotype analysis. Learn how to perform absolute cell counts and prepare samples for analysis.

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Hematopoietic Stem/Progenitor Cells CFC Assays & Immunophenotype

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  1. Hematopoietic Stem/Progenitor CellsCFC Assays & Immunophenotype C. Carlo-Stella, M.D. “Cristina Gandini” Medical Oncology Unit Istituto Nazionale Tumori and University of Milano, Italy

  2. Hematopoietic Stem Cells • Regulate the production, maintenance and regeneration of the many different myelo-lymphoid blood cell types throughout life span • HSCs are regulated by cell-autonomus and cell-nonautonomous mechanisms quantitative matter: in humans, HSCs produce 70 kg of cells per year

  3. Stem and Progenitor Cells HSC CMP CLP MEP GMP T B Mo Gr Me Er

  4. SPLEEN COLONY Till and McCulloch, Radiat Res 1961 Clonal Origin of Hematopoiesis • SELF-RENEWAL • DIFFERENTIATION • PROLIFERATION Spleen colony assay = the first quantitative test for stem cells

  5. Hematopoietic Progenitor Cells

  6. Heterogeneity of Human HSCs

  7. CD34+ CD133+ Thy1+ CD38- CD117- Lin- None of the markers are tied to unique stem cell functions or truly define the stem cell Cell Surface Markers of HSCs

  8. CD34+ Cells

  9. Analisi dei Progenitori Emopoietici Protocollo ISHAGE • Absolute cell count • Single platform • Lyse-no wash

  10. ABSOLUTE CELL COUNTS A KNOWN NUMBER OF FLUORESCENT MICROBEADS IS ADDED TO AN UNKNOWN NUMBER OF CELLS IN A KNOWN STARTING BLOOD VOLUME BLOOD CELLS AND BEADS EVENTS ARE ACQUIRED SIMULTANEOUSLY, AND THEIR RESPECTIVE REGIONS ARE IDENTIFIED. GATE 1 (Cells) Parameter 2 GATE 2 (Beads) THE ABSOLUTE CELL NUMBER IS CALCULATED ACCORDING TO THE Following FORMULA: Parameter 1 ABSOLUTE N°of CELL Events (Gate 1) N° of BEADS per X CELL = Unit of Volume COUNT N°of BEAD Events (Gate 2)

  11. Preparazione del Campione (1) RACCOLTA del CAMPIONE • Il sangue periferico deve essere raccolto in provette con EDTA • I campioni di Leucaferesi vengono raccolti utilizzando ACD come anticoagulante • Il campione deve essere mantenuto a temperatura ambiente prima della colorazione • Possibilmente la colorazione deve avvenire entro 12 ore dalla raccolta

  12. Preparazione del Campione (2) MATERIALI e REAGENTI • CD45 FITC e CD34PE • Cloruro di Ammonio • PBS (pH 7,2 + 0,1% sodiazide) con 2% FBS • 7 Amino Actinomicina D (7-AAD) per valutazione vitalità • Provette BD TruCount™

  13. Conta delle Cellule CD34+ con Provette TRUCOUNT™ ALLESTIRE IL CAMPIONE IN DUPLICATO 20 µL CD45FITC/CD34PE +20 µL 7-AAD + 100 µL Sangue Diluire il SANGUE PERIFERICO o la LEUCAFERESI < 20,000 WBC/µL VORTEX Incubazione a +4°C al buio 20 minuti 2 ml Cloruro di Ammonio LISI Incubazione T.A. al buio 10-15 minuti Acquisire entro un’ora dalla lisi

  14. “Thresold” per eliminare detriti “Thresold” su FSC scomparsa delle BEADS ! “Thresold” su FL1

  15. Selezione delle TruCount “beads” Attenzione alla eventuale interferenza fra TruCount ed eventi CD34+

  16. Acquisizione con CellQUEST sequenza della procedura detriti gate di pulizia

  17. G1 = R1 and not R6 G2 = R1 and R2 G3 = G2 and R3 CD34+ = R1 + R2 + R3 + R4 NON R6 e R9 G9 (cellule morte) = R1 and R9 and not R6

  18. 125 x 50147 x 2 = 50 /µL 2507100 CD34 x beads in tube x 2 = CD34+/µL Beadssangue

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