Flow-cytometry-based protocols for human blood/marrow immunophenotyping with minimal sample perturbation

Abstract

Summary This protocol provides instructions to improve flow cytometry analysis of marrow/peripheral blood cells by avoiding erythrolytic solutions, density gradients, and washing steps. We describe two basic approaches for identifying cell surface antigens with minimal sample perturbation, which have been successfully used to identify healthy and pathologically rare cells. The greatest advantage of these approaches is that they minimize the unwanted effect caused by sample preparation, allowing for improved study of live cells at the point of analysis. For complete details on the use and execution of this protocol, please refer to Petriz et al. (2018) .

Erythrocyte lysing solutions have a detrimental effect in flow cytometric dendritic cell detection

“…Minimal sample perturbation methods used in this study were previously described by our group [34,35,45] and one approach consists of discriminating nucleated cells from erythrocytes using viable permeant DNA-binding stains. Briefly, blood samples are incubated with a DNA stain and with antibodies directed against the markers of choice, avoiding the use of lysing and washing steps.…”

Protocol for generating human immune system mice and hydrodynamic injection to analyze human hematopoiesis in vivo

“… Acquire the samples to collect the desired volume of sample using available flow cytometer. Note: For detailed guidelines and description of flow cytometer set-up when performing flow cytometry experiments please refer to ( Cossarizza et al., 2021 ; Prosser et al., 2021 ; Rico et al., 2021 ) Analyze FCS files using flow cytometry analysis software. We routinely use NovoExpress Software or FlowJo for flow cytometer data analysis (a gating strategy is shown in Figure 4 ).…”

“…Note: For detailed guidelines and description of flow cytometer set-up when performing flow cytometry experiments please refer to ( Cossarizza et al., 2021 ; Prosser et al., 2021 ; Rico et al., 2021 )…”

Accurate identification of cell doublet profiles: Comparison of light scattering with fluorescence measurement techniques

“…Although DCV can be used for cell cycle and ploidy calculations using a linear scale, when unlysed blood is analyzed, Log scale allows easier exclusion of necrotic cells that can be present in the sample by compressing the scale to visualize strong signals [14]. Moreover, many permeant stains can be effluxed by multidrug transporters, which can be problematic for both analytical and sorting purposes.…”