Supplementary MaterialsImage_1. G-CSF treatment remains insufficiently comprehended. We tested the functionality of neutrophils isolated from Afatinib tyrosianse inhibitor blood of G-CSF-treated healthy SC donors. We quantified chemotaxis, oxidative burst, and phagocytosis before and after treatment and detected significantly reduced chemotactic activity upon G-CSF treatment. Similarly, treatment of previously untreated neutrophils with G-CSF led to reduced chemotactic activity. In addition, we revealed that this effect persists in the allogeneic SC recipients up to 4 weeks after neutrophil engraftment. Our data indicates that neutrophil quantity, as a single measure of immunocompetence in high-risk patients should be considered cautiously as neutrophil functionality might be affected by the primary treatment. = 11)= 12)leukemia= 3Aadorable lymphocytic leukemia= 2Myelofibrosis= 2Myelodysplastic syndrome= 2Chronic myelogenous leukemia= 1Hemophagocytic lymphohistiocytosis= 1 Open in a separate windows and G-CSF treatment and neutrophils were isolated using the same isolation process. In addition, we used blood from healthy volunteers not involved in any clinical treatment for cell activation analyses. Neutrophil isolation and maturity assessment Peripheral venous blood was collected in EDTA-containing tubes and neutrophils were isolated using a Histopaque-1119 separation followed by a Percoll gradient, as previously explained (25, 26). Viable cells were counted in a Neubauer chamber using trypan blue staining and subsequently diluted according to assay protocols. As exhibited earlier, this procedure prospects to a neutrophils purity of 93% (27). To assess neutrophil maturity, full blood as well as isolated neutrophil fractions were analyzed for their exact cell composition. We prepared blood smears on glass slides as well as smears from neutrophil isolations, which were resuspended in plasma from your same sample. These smears were fixed and Giemsa stained. Trained personnel used these smears to perform a comprehensive blood differentiation analysis at the Unit of Laboratory Medicine at the Norrland University or college Hospital in Ume? (project number FFLA01). All samples were analyzed in a blinded fashion. Band cells were considered immature, cells made up of Afatinib tyrosianse inhibitor a segmented nucleus were considered mature (28, 29). Media and incubation If not stated normally, neutrophil functionality assays were performed in RPMI medium Afatinib tyrosianse inhibitor without phenol-red (Lonza) and incubation occurred under standard cell culture conditions: 37C, 5% CO2, humid atmosphere. Chemotaxis Neutrophils chemotaxis was quantified using a transwell setup similar to previous description (30, 31). Briefly, 5 106 neutrophils/ml were stained with 3.3 M bis-2-carboxyethyl-5-[and-6]-carboxyfluorescein-AM (Sigma-Aldrich) for 20 min at room temperature, washed, and resuspended in medium with 0.05% human serum albumin. Fluorescence-impermeable transwell inserts (BD Falcon, HTS FluoroBlok, 3 m pore size, PET membrane) were loaded with 5 105 cells each and placed into wells of 24-well plates made up of 600 l RPMI, with or without 10 nM formyl-methionyl-leucyl-phenylalanine (fMLF). Fluorescence intensity (FI) with excitation 485 nm and emission 520 nm was detected in a plate reader (FLUOstar OMEGA; BMG Labtech) with 5% CO2 and 37C over the course of 30 min. Medium only served as blank and 5 105 stained cells seeded directly into the lower compartment served as 100% FI reference. If indicated, we added 10 ng/ml or 50 ng/ml G-CSF (human recombinant G-CSF, Sigma-Aldrich) to the respective neutrophil sample. Subsequently, cells were washed in medium and used as explained. Thus, if not stated otherwise, neutrophils are unstimulated prior to the analysis. Migration was calculated as percent of 100% reference Migration of unstimulated cells was subtracted from your fMLF-induced migration. Half migration time was decided as time required for 50% of maximum migration. Velocity at half migration time was calculated as %/min (observe Physique S2). Reactive oxygen species production Oxidative burst was quantified by luminol-based chemiluminescence (26). Briefly, 5 104 neutrophils per well were seeded in white 96-well plates with 50 M luminol and 1.2 U/ml horseradish peroxidase (both Sigma-Aldrich). Neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) or left untreated. Chemiluminescence as a measure of hydrogen peroxide production was detected every 2 min for 3 h in an Infinite 200 luminometer (Tecan). ROS were quantified by the area under the curve (AUC) calculated for the time given (3 h) and reduced by background of unstimulated cells. Phagocytosis Phagocytosis was assessed using pH rodo beads (pHrodoTM Red bioparticles conjugated for phagocytosis, Lifetechnologies) that switch their FI pH-dependently. Briefly, 5 104 neutrophils per well were added into a black 96-well plate with 100 g beads/well. Beads put directly into a buffer with pH 4 served as 100% FI control. Beads in medium with pH 7.4 served as blank. Neutrophils pre-treated with 10 M cytochalasin D for 15 min to block phagocytosis served as unfavorable control. FI Rabbit Polyclonal to OR8J3 (540/580 nm) was Afatinib tyrosianse inhibitor measured every 60 min for 3 h (FLUOstar Omega plate reader, BMG Labtech). Plasma.