cell culture human monocytic u937 cells  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition

doi: 10.3390/ijms25147917

Figure Lengend Snippet: Characterization of HNE-like activity in mature human neutrophils and human myeloid precursor cell lines. ( A ) HNE-like activity was measured in cell lysates of isolated peripheral PMN and the cell lines U937, HL-60, and HMC-1 using the fluorogenic substrate MeOSuc-AAPV-AMC. HNE-like activity is expressed in nM AMC/min/10 6 cells based on an AMC standard curve. Both mean ± SEM and individual values from 3 individual experiments with three technical replicates each are shown; ***, p ≤ 0.0001 to all other groups. ( B ) HNE-like activity in cell lysates is measured compared to isolated HNE (0.5 nM) after incubation (1 h, 37 °C) with buffer, the serine protease inhibitor Pefabloc SC (1 mM), or the elastase-specific inhibitor sivelestat (4 µM), or after one freeze/thawing-cycle. Data are mean ± SEM, n ≥ 3; ***, p ≤ 0.0001; ns, no inhibition. ( C ) Detection of HNE by western blot in lysates of human PMN (100,000 cells/lane), U937, HL 60, and HMC-1 cells (500,000 cells/lane each). 10 and 50 ng isolated HNE were used as positive controls. The blot is representative of 3 independent experiments.

Article Snippet: Cell culture media for U937 cells (RPMI 1640 with L-Glutamine and Sodium Bicarbonate, R8758) and for HL-60 and HMC-1 cells (Iscove’s Modified Dulbecco’s Medium (IMDM) with L-Glutamine, FG0465) were from Sigma-Aldrich (Taufkirchen, Germany), and fetal bovine serum (FCS) Gold (A15-751) from PAA Laboratories (Pasching, Austria).

Techniques: Activity Assay, Isolation, Incubation, Protease Inhibitor, Inhibition, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition

doi: 10.3390/ijms25147917

Figure Lengend Snippet: Kinetic parameters K i and IC 50(60min) for the reversible and irreversible inhibitors, respectively, were used in this study to profile isolated HNE and HNE in U937 lysates. Isolated HNE and U937 lysates were incubated with increasing inhibitor concentrations for one hour, and residual HNE activity was measured after the addition of substrate. The inhibition curves were then fitted to Morrisons’s equation and to a logistic equation to obtain K i and IC 50(60min) , respectively. Values are mean ± SD, n ≥ 3 individual experiments.

Article Snippet: Cell culture media for U937 cells (RPMI 1640 with L-Glutamine and Sodium Bicarbonate, R8758) and for HL-60 and HMC-1 cells (Iscove’s Modified Dulbecco’s Medium (IMDM) with L-Glutamine, FG0465) were from Sigma-Aldrich (Taufkirchen, Germany), and fetal bovine serum (FCS) Gold (A15-751) from PAA Laboratories (Pasching, Austria).

Techniques: Isolation, Incubation, Activity Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition

doi: 10.3390/ijms25147917

Figure Lengend Snippet: Effect of inhibitors on HNE in U937 cells as a model of immature neutrophils. Cells were cultured for 0–26 h with increasing concentrations of the compounds or solvent controls. Subsequently, cells were washed and lysed, and residual HNE activity was measured using the HNE substrate MeOSuc-AAPV-AMC. Residual activity is expressed as a percentage of matched solvent controls. Data are mean ± SEM, n ≥ 3; ***, p ≤ 0.0001; **, p ≤ 0.001; *, p ≤ 0.05; ns, not significant.

Article Snippet: Cell culture media for U937 cells (RPMI 1640 with L-Glutamine and Sodium Bicarbonate, R8758) and for HL-60 and HMC-1 cells (Iscove’s Modified Dulbecco’s Medium (IMDM) with L-Glutamine, FG0465) were from Sigma-Aldrich (Taufkirchen, Germany), and fetal bovine serum (FCS) Gold (A15-751) from PAA Laboratories (Pasching, Austria).

Techniques: Cell Culture, Solvent, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition

doi: 10.3390/ijms25147917

Figure Lengend Snippet: Effect of inhibitors on HNE in mature PMN. Freshly isolated PMN were incubated for 1 h with increasing concentrations of the inhibitors or solvent controls. Subsequently, cells were washed and lysed, and residual HNE activity was measured using the HNE substrate MeOSuc-AAPV-AMC. Residual activity is expressed as a percentage of matched solvent controls. Data are mean ± SEM, n ≥ 3; ***, p ≤ 0.0001; **, p ≤ 0.001; ns, not significant. For comparisons, the effects of the inhibitors on U937 cells (0–3 h, see ) are shown in different shades of blue.

Article Snippet: Cell culture media for U937 cells (RPMI 1640 with L-Glutamine and Sodium Bicarbonate, R8758) and for HL-60 and HMC-1 cells (Iscove’s Modified Dulbecco’s Medium (IMDM) with L-Glutamine, FG0465) were from Sigma-Aldrich (Taufkirchen, Germany), and fetal bovine serum (FCS) Gold (A15-751) from PAA Laboratories (Pasching, Austria).

Techniques: Isolation, Incubation, Solvent, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition

doi: 10.3390/ijms25147917

Figure Lengend Snippet: Kinetics of the inhibition of HNE. The association rate k on of the formation of the HNE/inhibitor complex was determined using pre-steady-state kinetics. The dissociation rate k off of complex with a reversible inhibitor is derived from the k on and the K i determined using steady-state kinetics (see Table 1 ). From these values, the half-time of the complex formation t 1/2 , on at the maximal inhibitor concentration Imax applied to U937 cells, and of its dissociation are calculated. Values are mean ± SD, n ≥ 3. * Data from [ 35 ], as the reaction with alvelestat is too fast to be recorded precisely with our setup.

Article Snippet: Cell culture media for U937 cells (RPMI 1640 with L-Glutamine and Sodium Bicarbonate, R8758) and for HL-60 and HMC-1 cells (Iscove’s Modified Dulbecco’s Medium (IMDM) with L-Glutamine, FG0465) were from Sigma-Aldrich (Taufkirchen, Germany), and fetal bovine serum (FCS) Gold (A15-751) from PAA Laboratories (Pasching, Austria).

Techniques: Inhibition, Derivative Assay, Concentration Assay