12/31/2023 0 Comments Facs analysis![]() As above, SP KLS were sorted into carrier cells and then both populations were stained for Pyronin Y analysis. (B) Analysis of HSC cell cycle status by Pyronin Y staining. This plot allows discrimination of the various stages of cell cycle of the HSC population. On the reanalysis, HSCs (Sca-1 +B220 −) were easily identified from carrier cells (Sca-1 −B220 +) and gated to show a distribution of Ki-67 versus DNA content using propidium iodide. (A) Using the carrier cell technique, SP KLS cells were purified then fixed and permeabilized for Ki-67 staining. If we were analyzing transplanted mice in which the GFP was not used to follow retroviral marking, we typically use CD45.1-FITC to track the recipient cells in addition to the donor cells.Ĭell cycle reanalysis of purified SP KLS cells by Ki-67 and Pyronin Y. The B cells are the double positive population, the myeloid cells (Gr-1 +, Mac-1 +) are the PeCy7 +PacBlue − population, while the T cells (CD4 +, CD8 +) are the PeCy7 − PacBlue + population. By labeling B cells with both B220-PeCy7 and B220-Pac-Blue, all major hematopoietic lineages can be displayed simultaneously on the same plot. T-cells are labeled with CD4 and CD8 antibodies conjugated to Pacific Blue (Pac-Blue). Myeloid cells are labeled with Gr1 and Mac-1 conjugated to PeCy7. The CD45.2 + / GFP + population can then be gated to a PeCy7 versus Pacific Blue dot-plot to analyze distribution of the blood lineages. Progeny of donor HSC (CD45.2) can be discriminated from recipient cells (CD45.1) by CD45 alleles, and the donor HSCs that were successfully transduced to over-express a test gene are GFP +. Then viable white blood cells are gated and displayed on a CD45.2-APC versus GFP dot-plot. For this analysis, red blood cells are depleted or lysed, and the remainder gated out on a FSC/SSC plot. Rapid flow cytometric analysis of peripheral blood of mice transplanted with HSCs transduced with MSCV-GFP retrovirus. The purpose of updating this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.Ĭopyright © 2012 International Society for Advancement of Cytometry. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. In this updated version of our review from 2009, we review different strategies for hematopoietic stem and progenitor cell identification and purification. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. The frequency of HSCs in murine bone marrow is about 0.01% of total nucleated cells and ∼5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. However, despite over 40 years of research, working with HSCs in the mouse remains challenging because of the relative abundance (or lack thereof) of these cells in the bone marrow. We are experienced developers and decided to start working on our own analysis program, with the goal to make it free and easy to use for all.įlow cytometry is such an important and ubiquitous technique with many different applications.Hematopoietic stem cells (HSCs) remain the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. When we changed computer systems the software we had been using was not compatible with our new system. We had been using proprietary commercial flow cytometry analysis software. Experienced low-level C programmers who are dismayed with the inefficiency of modern day enterprise and web software.Knowledgable in basic flow cytometry and analysis.Experts in advanced flow cytometry techniques.Linear, Log, Logicle, Hyperlog transforms.Rectangle, Polygon, Range, Ellipse, Quadrant, and Boolean Gates.Dot Plots, Histograms, Histogram Overlays.We work together on floreada.io over the internet with the goal to create such software.įloreada.io is new and we are adding additional features all the time. We all have different backgrounds and are from different institutions (or no institution at all), but we are united in the belief that there should be easy-to-use, free flow cytometry software that is available to all. ![]() ![]() We are all programmers who have some kind of connection to flow cytometry and are frustrated with aspects of current analysis software (high cost, no Linux support, limitations on the number of computers it can be used on, need to use a dongle). ![]()
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