Anti cd45 apc h7

For further purification, MNCs from freshly resected patient tissue specimens were subjected to Ficoll‐Hypaque gradient centrifugation for 30 min at 24°C. Next, the MNC layer was transferred to a new tube, washed twice with phosphate-buffered saline (PBS), and suspended in PBS. ILCs were sorted using a BD FACSAria system (BD Bioscience) as Lin⁻-enriched MNCs as Lin cocktail⁻ (CD3, CD19, CD20, and CD14), CD94⁻ CD34⁻ CD1a⁻ TCRα/β⁻ TCRγ/δ⁻ CD45⁺ CD127⁺ CRTH2^+/− CD117^+/− cells using FITC anti-Lin (643510; BD Bioscience), CD94 (305504; Biolegend, San Diego, CA, USA), CD34 (343504; Biolegend), CD1a (300104; Biolegend), T cell receptor (TCR)α/β (306706; Biolegend), TCRγ/δ (331208; Biolegend), allophycocyanin (APC)-H7 anti-CD45 (56017; Biolegend), Percp-cy5.5 anti-CD127 (351322; Biolegend), phycoerythrin (PE)-Cy7 anti-CRTH2 (350118; Biolegend), and BV605 anti-CD117 (562687; Biolegend). pDCs were sorted as Lin⁻ CD94⁻ CD34⁻ CD1a⁻ TCRα/β⁻ TCRγ/δ⁻ CD45⁺ BDCA2⁺ cells using FITC anti-Lin, CD94, CD34, CD1a, TCRα/β, TCRγ/δ, APC-H7 anti-CD45, and APC anti-BDCA2 (17-9818-42; Biolegend). Purity was routinely >99%. Cell viability was determined by trypan blue staining and was >99% after isolation.

Samples were collected either in Transfix tubes (MBL International) or EDTA tubes. Cells from EDTA tubes were isolated by Ficoll-Paque or RBC lysis. Cells were then fixed by incubating in Fixation Buffer (BD Cytofix,) at 4 °C for 20 min. Transfix samples were processed as per the manufacturer’s instructions. Fixed cells were labeled with cell surface markers, anti-CD45 APC-H7, anti-CD33 PerCP/Cy5.5, anti-CD123 BV650 (BioLegend, clone 6H6, cat. 306020), anti-CD38 PE/Cy7(BioLegend, clone HIT2, cat. 303516), anti-CD34 PE-CF594 (BD Biosciences, clone 581, cat. 562383), and anti-CD3 BV605 (BioLegend, clone SK7, cat. 344836). Subsequently, cells were permeabilized by incubating in Perm/Wash buffer (BD Biosciences) at 4 °C for 30 min. After washing, cells were labeled with anti-phospho-STAT5 AlexaFluor 647 (Y694), anti-phospho-ERK1/2 AlexaFluor 488 (T202/Y204), phospho-S6 V450 (S235/S236) and anti-phospho-SYK PE (Y525/526,) at 4 °C for 15 min. Flow cytometry evaluation was performed on BD LSRFortessa. Data were analyzed using FlowJo software.

PU-FITC-binding assay was performed as described before^{1 (link),8 (link)}. Evaluation for epichaperome abundance was performed on samples obtained prior treatment with PU-H71, except where indicated (i.e., Fig. 3e, f.) Briefly, isolated cells were incubated with PU-FITC that binds to epichaperome, or FITC9 (FITC control that does not bind to the epichaperome) at 37 °C for 4 h. After washing out PU-FITC or FITC9, cells were labeled with anti-CD45 APC-H7, anti-CD33 PerCP/Cy5.5 (BioLegend, clone WM53, cat. 303414) anti-CD3 PE, and anti-CD34 APC to distinguish subpopulations. Intensity of FITC of each subpopulation was measured by flow cytometry on BD LSRFortessa (BD Biosciences). Data was analyzed using FlowJo software (BD). The epichaperome abundance was evaluated by calculating relative intensity of FITC after correcting the background from FITC9 of each subpopulation to that on T-lymphocytes (CD45^highCD3 + ) harboring low abundance of epichaperome (MFI of target population/MFI of T-lymphocytes)^{8 (link)}.