SMCs-Vascular smooth muscle cells were counted manually (camera) and 20,000 cells were plated in a 12 multiwell plate dishes (Thermo scientific). Cells were kept in Opti-MEM medium (Gibco), 24 and 48 hours post plating, and were then counted using Beckman Z1 coulter particle counter. ECs-HUVEC were seeded on 24-multiwell plate coated with PBS-GEL at 10,000 cells/well in medium supplemented with 20% serum and allowed to attach for 4-5 hours. Cells were changed to media with 10% serum supplemented with VEGF, VEGF + βAPN or VEGF + βAPN + TGF-β and Negative control (no treatment). Cells were allowed to proliferate for 48 hours. Treatments were replaced on a daily basis. Cell number was determined by Beckman Z1 coulter particle counter. Following similar treatments for 48 hours, cells were fixed and stained using anti-Ki67 to directly monitor proliferating cells. Relative proliferation was measured using the ratio between Ki67 expressing nuclei and number of cells.
Beckman z1 coulter particle counter
Manufactured by Beckman Coulter
Sourced in United States
The Beckman Z1 Coulter® Particle Counter is a laboratory instrument designed to measure and count the size and number of particles suspended in a fluid sample. It operates on the principle of electronic impedance to detect and enumerate particles passing through a small aperture.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (2 mentions)
Collagenase a, by Roche (2 mentions)
U bottom plate, by Greiner (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Rpmi 1640 medium, by Lonza (2 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
70 m nylon cell strainer, by BD (1 mentions)
Rpmi 1640 culture medium, by Lonza (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
4 protocols using beckman z1 coulter particle counter
1
Quantifying Vascular Cell Proliferation
2
Isolation and culture of lung cells
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Lung cell suspensions were prepared by cutting the lung into small pieces and by adding digestion buffer, containing DNase I and Collagenase A (Roche Diagnostics, Germany), for 30 min. The digestion was stopped using fetal calf serum (FCS; Hyclone Laboratories, USA). The lung pieces were transferred toward a 70-µm nylon cell strainer (BD Biosciences, The Netherlands) and rinsed with 10 mL RPMI. Cells were washed and resuspended in RPMI-1640 culture medium (Lonza, USA) supplemented with 10 % heat-inactivated FCS and 0.1 % penicillin–streptomycin solution (Sigma-Aldrich). Total number of cells was calculated using a Beckman Z1 coulter® Particle Counter (Beckman, USA). Lung cells (4 × 105 cells/well) were cultured in a Greiner bio-one CellSTAR 96-well U-bottom plate (Greiner Bio-One B. V., The Netherlands) in medium with or without 50 µg/mL HDM (Greer Laboratories, USA). The supernatant was harvested after 4 days of culture at 37 °C in 5 % CO2 and stored at −20 °C until further analysis [13 (link)].
3
Splenocyte isolation and cytokine analysis
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Fresh spleens were collected aseptically and homogenized using the top of a syringe to gently pass through a 70 μm nylon cell strainer. The collected cell suspension was washed and incubated with lysis buffer (Thermo Fisher, Netherlands), for 5 min to remove red blood cells. Splenocytes were washed and resuspended in RPMI 1640 medium (Lonza), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Bodinco, Alkmaar, Netherlands), penicillin (100 U/mL)/streptomycin (100 μg/mL; Sigma-Aldrich) and β-mercaptoethanol (20 μM; Thermo Fisher Scientific). Total cell number was determined by using a Beckman Z1 coulter® Particle Counter (Beckman, United States) and splenocytes (107 cells/ml) were cultured in 96-well U-bottom plates (Greiner Bio-One B. V., Netherlands), either with medium or with 0.2 μg/mL anti-CD3 (Thermo Fisher, Netherlands). After 4 days stimulation, the supernatants were harvested and stored at -20°C until further analysis. The concentrations of MCP-1, IL-17A, IL-13, IL-22, and IFN-γ were measured using Bead-based immunoassays (Procartaplex, Thermo Fisher, Netherlands) according to the manufacturer’s instructions.
4
Lung Cell Isolation and Culture
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Fresh lungs were collected aseptically, cut into small pieces and homogenized using enzymatic digestion buffer (DNase I and Collagenase A, Roche Diagnostics). After 30 min incubation, the enzymatic digestion process was stopped by adding fetal calf serum (FBS, Bodinco, The Netherlands). Lung pieces were gently passed through a 70 µm nylon cell strainer. The collected cell suspensions were washed and resuspended in RPMI 1640 medium (Lonza, The Netherlands) and supplemented with 10% heat-inactivated FBS and penicillin (100 U/mL)/streptomycin (100 µg/mL; Sigma-Aldrich). Total cell number was determined by using a Beckman Z1 coulter® Particle Counter (Beckman, USA) and lung cells (106 cells/ml) were cultured in 96-well U-bottom plates (Greiner Bio-One B. V., The Netherlands), either with medium or with medium supplemented with 50 μg/mL HDM (Greer Laboratories). After 4 days of culture at 37°C in 5% CO2, cell culture supernatant was collected and stored at −20°C until further analysis.
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