Flow jo version 7

Manufactured by Tree Star

Sourced in United States

FlowJo version 7.0 is a software application that provides comprehensive analysis of flow cytometry data. It offers tools for data visualization, gating, and statistical analysis, enabling users to efficiently process and interpret their flow cytometry experiments.

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5 protocols using flow jo version 7

Isolation and Identification of Immune Cells

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Single cells were harvested using 45 μm filters (Biologix, Shanghai, China) from spleen and draining lymph nodes at the peak of psoriasis. At first, cells were washed three times with PBS. Surface staining of innate immune cells was performed with fluorescent-labeled antibodies: F4/80-PerCP-Cy 5.5, CD11c-PE, CD11b-APC, and GR-1-FITC. Staining of γδ T cells and their subsets was performed with CD3-APC, γδ TCR-PE, TCR-β-BV421, Vγ4-FITC, and Vγ5-BV510 (BD Biosciences, New Jersey, USA) antibodies for 30 min at RT. FACS was done using LSR II (BD Biosciences) and data were analyzed using Flow Jo version 7.0 (Tree Star, California, USA).

Wu H., Ou J., Li K., Wang T, & Nandakumar K.S. (2023). Comparative studies on mannan and imiquimod induced experimental plaque psoriasis inflammation in inbred mice. Clinical and Experimental Immunology, 211(3), 288-300.

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Isolation and Phenotyping of Skin and Immune Cells

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Skin samples were separated into dermis and epidermis layers after digestion with dispase II (10 mg/ml, Solarbio, Beijing, China) for 2 h at 37°C. To obtain single cells, shredded dermal tissue was treated with collagenase II (3 mg/ml, Solarbio) and DNase I (5 mg/ml, Solarbio) at 37°C for 120 min. Single cells were also prepared from inguinal lymph nodes and spleen from mice by maceration. Surface staining was performed with fluorescent-labeled antibodies F4/80-PerCP-Cy 5.5, CD11c-PE, CD11b-APC, Ly6C/6G-FITC, CD45-PerCP-Cy 5.5, γδ T-PE (BD Biosciences, New Jersey) for 30 min at room temperature. FACS was performed using LSR II (BD Biosciences) and data were analyzed using Flow Jo version 7.0 (Tree Star, California). Gating strategy for different cell populations was given in Supplementary Figure S3.

Wu H., Zeng L., Ou J., Wang T., Chen Y, & Nandakumar K.S. (2022). Estrogen Acts Through Estrogen Receptor-β to Promote Mannan-Induced Psoriasis-Like Skin Inflammation. Frontiers in Immunology, 13, 818173.

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Cell Cycle Analysis by Flow Cytometry

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In the present study, for the analysis of DNA content only, the harvested cells were fixed in 70% ethanol and stained with 50 µg/ml PI. The DNA content was analyzed by flow cytometric analysis (FACScan). For the determination of DNA and RNA content, in order to distinguish G0 from G1 cells, the harvested cells were fixed and permeabilized using a BD IntraSure kit, and subsequently intracellularly stained with an antibody against Ki-67 and with Hoechst 33342 (both from BD Bioscience). Data analysis was performed using FlowJo version 7.0 (TreeStar, Ashland, OR, USA).

Liu N., Wang C., Wang L., Gao L., Cheng H., Tang G., Hu X, & Wang J. (2016). Valproic acid enhances the antileukemic effect of cytarabine by triggering cell apoptosis. International journal of molecular medicine, 37(6).

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Quantification of CD71+ and CD117+ Cells

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The frequency of CD71⁺ and CD117⁺ cells was quantified in unfractionated peripheral blood samples using an established, single-platform enumeration method. Briefly, 100 μL of peripheral blood (one million cells) was labeled with 10 μL of BD PharmingenTM mouse antirat CD71 antibody conjugated with fluorescein isothiocyanate and 10 μL of BD PharmingenTM rat antimouse CD117 (c-Kit) antibody conjugated to phycoerythrin (BD Biosciences, Franklin Lakes, NJ) for 30 minutes. Following erythrocyte lysis, cells were then centrifuged at 300 G for 5 minutes, and the supernatant was discarded. Cells were washed three times and fixed with BD CytofixTM solution (BD). Cells were analyzed using BD FACSCalibur flow cytometer (BD) equipped with CellQuest software (BD). Samples from each group were stained and run in duplicate, and an event count of 30,000 was obtained for each run. Following acquisition of data, further analysis was performed using Flow Jo version 7.2.4 (Tree Star, Ashland, OR).

Pasupuleti L.V., Cook K.M., Sifri Z.C., Alzate W.D., Livingston D.H, & Mohr A.M. (2014). Do all β-blockers attenuate the excess hematopoietic progenitor cell mobilization from the bone marrow following trauma/hemorrhagic shock?. The journal of trauma and acute care surgery, 76(4), 970-975.

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Hoechst 33342 Cell Cycle Analysis

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On day 3 post transfection cells were trypsinized and resuspended in culture medium. 2 μg/mL of Hoechst 33342 (Thermo Scientific) were added, and the cell suspension was incubated for 20 min at 37°C. 1×10⁵ cells per sample were analyzed by flow cytometry at excitation/emission = 346/497 nm on LSR II (Becton Dickinson, Franklin Lakes, USA). The data were processed by FlowJo Version 7.2.4 (Tree Star, Ashland, USA) and Dean-Jett-Fox model was applied. Results are presented as a mean fold-change ± SD of at least three independent experiments.

Serguienko A., Grad I., Wennerstrøm A.B., Meza-Zepeda L.A., Thiede B., Stratford E.W., Myklebost O, & Munthe E. (2014). Metabolic reprogramming of metastatic breast cancer and melanoma by let-7a microRNA. Oncotarget, 6(4), 2451-2465.

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