celltrace cfse cell proliferation kit life cat  (Dojindo Labs)

Journal: bioRxiv

Article Title: Ferritinophagy is a Druggable Vulnerability of Quiescent Leukemic Stem Cells

doi: 10.1101/2023.12.18.572101

Figure Lengend Snippet: A. Serial transplantation of primary AML specimens into immunodeficient NSG mice established a substantial reservoir of leukemia cells, enabling a wide range of assays, including in vivo cell kinetic experiments. B. Cell cycle analysis of human CD45 + leukemic cells was conducted using DAPI and staining with IgG (isotype) or Ki67. C. Distribution of cells within the G 0 , G 1 , and S-G 2 -M phases was determined in patient-derived xenograft (PDX) assays (n=5). D. Mice harboring AML PDXs were treated with 100mg/kg BrdU twice a day via intraperitoneal injections for seven consecutive days. BrdU incorporation was evaluated by staining CD45 + human cells isolated from the mice’s bone marrow with an anti-BrdU antibody. Quantification of BrdU incorporation was performed in distinct subpopulations based on their cell cycle status, including quiescent (Ki67 - ) and cycling cells (Ki67 + ). E. Representative contour plots illustrate BrdU labeling in quiescent or cycling human hCD45 + AML cells. F. The percentage of BrdU incorporation in quiescent versus cycling subpopulations was determined in vivo (n=6-7 mice per group in three different PDX assays). G-H . Human leukemic cells were stained with CD34 and CD38 antibodies. The proportion of quiescent ( G ) and BrdU + ( H ) cells was examined within each subpopulation defined by CD34/CD38 staining (n=7 mice per group in one PDX assay). I-J. Cycling or quiescent populations were sorted using pyronin Y and Hoechst and subsequently transplanted separately into NSG mice. The schematic representation of the experimental setup is shown ( I ). J. Engraftment of hCD33 + hCD45 + human leukemic cells was assessed after 8-12 weeks (n=4 PDX), and each dot represents the relative engraftment in each mouse. K-N. To assess the role of cycling and quiescent cells in AML propagation, PDX cells labeled ex vivo with CFSE were injected into NSG mice, and CFSE retention was measured by flow cytometry after one week. The experimental design is represented schematically ( K ). The CFSE intensity ( L ) and percentage ( M ) were quantified in the quiescent and cycling groups. Post-hoc analysis of CFSE retention was performed in Ki67 - and Ki67 + subpopulations of leukemic cells from group of mice transplanted with the cycling or quiescent fraction ( N ). The standard deviations are indicated by the vertical bars. The significance levels are denoted by ns (not significant), *p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: Dyes used were: DAPI Axonlab Cat#A4099.0, CellTrace CFSE Cell Proliferation Kit (Life Techniologies, Cat#C34570), Pyronin Y (Sigma Aldrich, Cat#P9172), Hoechst 33342 (Thermo Fisher Scientific, Cat#62249), 5-Bromo-2′-deoxyuridine (Sigma Aldrich, Cat#B5002), Propidium iodine (BD Biosciences, Cat#556463, RRID:AB_2869075), Cyto-ID (Enzo Life Sciences, Cat#ENZ-51031), BioTracker Labile Fe 2+ (Merck Millipore, Cat#SCT037), Transferrin From Human Serum, Alexa FluorTM 488 Conjugate (Thermo Fisher Scientific, Cat#T13342), Annexin V (BD Biosciences, Cat#556419, RRID:AB_2665412) and DRAQ7 + (Thermo Fisher Scientific, Cat#D15106).

Techniques: Transplantation Assay, In Vivo, Cell Cycle Assay, Staining, Derivative Assay, BrdU Incorporation Assay, Isolation, Labeling, Ex Vivo, Injection, Flow Cytometry