Phosflow lyse fix buffer

For analysis of peripheral blood lymphocyte populations, EDTA‐treated whole blood of HAM/TSP patients and healthy volunteers (HVs) were stained with CD3, CD4, CD8, CD14, CD19, CD25, CD27, CD45, CD45RA, CD45RO, CD56, CD95, CD122 (clone Mikβ2 and Mikβ3), CD197 (CCR7), CD244 (all from BD Biosciences), CD279 (PD‐1; BioLegend), and Tim‐3 (R&D Biosystems). Mikβ1 and Mikβ3 identified noncompeting epitopes on the IL‐2/IL‐15Rβ, but Mikβ1 and Mikβ2 appeared to recognize the same epitope or very closely related epitopes on the β chain.21, 25 Tax 11‐19/HLA‐A201 tetramer was provided by National Institute of Allergy and Infectious Diseases Major Histocompatibility Complex Tetramer Core Facility. CMV pp65/HLA‐A201 tetramer (Beckman Coulter) was used as control. For detection of phosphorylated STAT5 (pSTAT5), EDTA‐treated whole blood were incubated for 15 min at 37°C and lysed with BD Phosflow^TM Lyse/Fix buffer (BD Biosciences). After washing and permeabilization with cold 90% methanol on ice for 30 min, the cells were stained with antibodies for CD3, CD4, CD8, and pSTAT5 (all from BD Biosciences). CD107a mobilization assay was performed as previously described.20 All flow cytometric analyses were performed using a FACSCalibur or LSR II (both from BD Biosciences). The data were analyzed using FlowJo 10.2 software (FlowJo LLC).

Neutrophils were stimulated as described above for the examination of neutrophil oxidative burst with the exception that the stimulation was stopped after 5 min at 37°C by fixation with pre-warmed BD Phosflow^TM Lyse/Fix Buffer at 37ºC for 10 min. Cells were permeabilized with BD Phosflow^TM Perm Buffer II on ice for 30 min and then stained with Alexa Fluor^® 488 conjugated anti-phospho SYK antibody (pY348) at RT for 1 hr before flow cytometry analysis. All the above reagents were purchased from BD Biosciences (San Jose, CA, USA).