Training Novices in Generation and Analysis of High‐Dimensional Human Cell Phospho‐Flow Cytometry Data

Abstract

This article presents a single experiment designed to introduce a trainee to multiple advanced bench and analysis techniques, including high‐dimensional cytometry, profiling cell signaling networks, functional assays with primary human tissue, and single‐cell analysis with machine learning tools. The trainee is expected to have only minimal laboratory experience and is not required to have any prior training in flow cytometry, immunology, or data science. This article aims to introduce the advanced research areas with a design that is robust enough that novice trainees will succeed, flexible enough to allow some project customization, and fundamental enough that the skills and knowledge gained will provide a template for future experiments. For advanced users, the updated phospho‐flow protocol and the established controls, best practices, and expected outcomes presented here also provide a framework for adapting these tools in new areas with unexplored biology. © 2020 by John Wiley & Sons, Inc.Basic Protocol: Phospho‐protein stimulation and mass cytometry data collectionSupport Protocol: Analysis of signaling mass cytometry data

Immune cell regulation in stem cell niche contacting glioblastomas

“…Phospho-specific mass cytometry was performed as previously described (46). Cryopreserved samples were thawed in a water bath as above.…”

“…Cryopreserved samples were rapidly thawed in a 37 o C water bath and resuspended in complete RPMI supplemented with 10% FBS and 50 units/mL of penicillin-streptomycin (Thermo Scientific HyClone). Cell suspensions were processed and stained as previously described (44,46). Briefly, cells were washed once with serum free RPMI and subsequently stained with 103 Rh Cell-ID Intercalator (Fluidigm) at a final concentration of 1 uM for 5 minutes at room temperature.…”

STAT1Gain-of-Function Variants Drive Altered T Cell Prevalence, Metabolism, and Heightened IL-6 Sensitivity

“…Cryopreserved samples were rapidly thawed in a 37 o C water bath and resuspended in complete RPMI supplemented with 10% FBS and 50 units/mL of penicillin-streptomycin (Thermo Scientific HyClone). Cell suspensions were processed and stained as previously described [14,44,45]. Briefly, cells were washed once with serum free RPMI and subsequently stained with 103 Rh Cell-ID Intercalator (Fluidigm) at a final concentration of 1 uM for 5 minutes at room temperature.…”

“…Before automated high-dimensional data analysis, the mass cytometry data were transformed with a cofactor of 5 using an inverse hyperbolic sine (arcsinh) function. Cell doublets were first excluded using Gaussian parameters (Center, Offset, Width, Residual) as reported [14]. Intact cells were gated based on DNA content ( 191 Ir and 193 Ir).…”

“…Given the limited characterization of the immune milieu in these patients, we used high-dimensional mass cytometry to comprehensively characterize the lymphoid and myeloid compartments in two patients with STAT1 GOF mutations. Using this approach, we were able to track major mononuclear cell types frequencies and activation states, including phosphorylation and metabolism [9][10][11][12][13][14], and cytokine responses in two STAT1 GOF patients. These studies demonstrate the effect of pSTAT1 perturbations on a global cellular level and identified the impact of STAT1 GOF in specific cell subsets.…”