Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA). Surface staining was performed in the dark for 30 minutes at 4 ☌ in staining buffer.
#Flowjo software tree star software
All data collection and sorting were performed using BD FACS Diva software (BD Biosciences) and data analyses were performed using FlowJo software (Tree Star, Ashland, OR, USA).įor all experiments, cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT.
The spleen FACS experiments were performed using a FACSAria II instrument (BD Immunocytometry Systems) equipped with 405 nm, 488 nm, or 633 nm lasers located at the University of Chicago Flow Cytometry Core Facility, Chicago, IL, USA. Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA)įor flow cytometry immunophenotyping experiments, cells were acquired on an LSR II cytometer (BD Immunocytometry Systems, San Jose, CA, USA) equipped with 405 nm, 488 nm, 561 nm, and 640 nm excitation lasers. FlowGM thus provides rapid high-dimensional analysis of cell phenotypes and is amenable to cohort studies.For all experiments, cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT. Cell numbers and coefficient of variation (CV) were similar between FlowGM and conventional gating for lymphocyte populations, but notably FlowGM provided improved discrimination of “hard-to-gate” monocyte and dendritic cell (DC) subsets.
#Flowjo software tree star manual
Cluster labels were integrated into FCS files, thus permitting comparisons to manual gating. Meta-clustering in a reference donor permitted automated identification of 24 cell types across four panels. A Gaussian Mixture Model (GMM)-based approach was utilized for initial clustering, with the number of clusters determined using Bayesian Information Criterion. Here, we report a computational pipeline, called FlowGM, which minimizes operator input, is insensitive to compensation settings, and can be adapted to different analytic panels. However, robust high-dimensional post-analytic strategies for automated data analysis in large numbers of donors are still lacking.
Multi-parametric flow cytometry is a key technology for characterization of immune cell phenotypes.
0 kommentar(er)
