The BAL samples from ‘COSMIC cohort’, ‘Smoke Expo cohort’, and ‘CYREBAC cohort’) as well as BW (‘COSMIC cohort’) were filtered through a Dacron net (Millipore®) and centrifuged (400×g for 10 min at 4°C) and the total cell counts and Trypan Blue exclusion were carried out. The cell-free BAL or BW samples were immediately frozen (−80°C) for subsequent analyses. We also prepared BAL cell cytospin slides for differential counts (stained with May–Grünwald and Giemsa) and for immunocytofluorescence (ICF) staining. Aliquoted cells were stored for further analysis. The bronchial tissue biopsies (COSMIC cohort) were immediately formalin-fixed and embedded in paraffin. The material was evaluated, and one representative tissue block from each case was selected for immunohistochemistry (IHC) analysis. For the IS from the BALO cohort, one aliquot from the COPD patients was processed in 0.65 mM DTT, filtered, and stored at –80°C for subsequent analyses.
Dacron net
Manufactured by Merck Group
Sourced in Ireland
Dacron net is a type of laboratory equipment made of polyester fibers. Its core function is to provide a sturdy and durable support structure for various laboratory applications.
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Lab products found in correlation
4 protocols using dacron net
1
Analyzing Cell Composition in Respiratory Samples
2
Bronchoalveolar Lavage Fluid Analysis
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BAL was performed under local anaesthesia by an experienced physician as previously described [16 (link)]. In short, a flexible fibreoptic bronchoscope was passed transorally and wedged into the middle-lobe bronchus. Sterile phosphate buffered saline at 37°C was instilled in five aliquots of 50 ml and immediately re-aspirated and collected into a siliconised plastic bottle kept on ice. The mean recovery of the instilled fluid was 63% (min-max range 42–73%). BALF was strained through a Dacron net (Millipore, Cork, Ireland) before centrifugation. The cell pellet was resuspended in cold phosphate buffered saline (PBS) pH 7.4, and kept on ice throughout the experiments. BAL fluid differential cell counts were based on May-Grünwald and Giemsa staining (Table 2 ).
3
Characterization of CD4+ Vα2.3+ T-cells in BALF
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The BAL was performed according to procedures earlier described [10 (link)]. The BALF was strained through a Dacron net (Millipore, Cork, Ireland) and centrifuged, thereafter the supernatants were removed. The cell pellet was resuspended in PBS and antibodies used as surface markers were added, and thereafter incubated at 4o C for 20 min. After incubation, cells were washed twice with cell wash (BD Bioscience, Mountain View, CA, U.S.A.). Surface markers expressed on T cells were analyzed with flow cytometry using FACS CANTO II flow cytometer (BD Bioscience). Data were processed using a FACS Diva 6.1.2 software (BD Bioscience). The antibody used as surface marker for CD4+ Vα2.3+ T-cells was Vα2.3 (Thermo Scientific), see Fig. 1 for a typical staining.
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An example showing a population of CD4+ Vα2.3+ T-cells in BALF
4
Isolation and Cultivation of PBMCs and BAL Cells from Sarcoidosis Patients
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Peripheral blood mononuclear cells (PBMCs) from sarcoidosis patients and healthy blood donors were freshly isolated from whole blood samples by density gradient centrifugation (Lymphoprep, Axis-Shield; Oslo, Norway). PBMCs were resuspended in complete medium (RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), L- glutamine, penicillin and streptomycin, all from Hyclone; Logan, UT) before use. When appropriate, PBMCs were frozen in 90% FBS+10% DMSO at -80 °C until laboratory analysis. BAL cells from sarcoidosis patients (n = 13) and healthy controls (n = 3), were obtained with bronchoscopy. The bronchoscopy procedure and handling of the BAL fluid was conducted as previously described [18] . In short, the BAL was performed in a sub-segmental bronchus in the middle lobe by instilling five aliquots (each 50 ml) of sterile, phosphate buffered saline solution. The fluid was gently and directly suctioned back, pooled and passed through a Dacron net (Millipore, Cork, Ireland). BAL cells were collected and resuspended in PBS after 10 + 10 min centrifugation (1400 rpm followed by 2000 rpm, 4 °C). Target cells used in the CTLassay, the murine lymphoblast-like mastocytoma cell line P815 (#TIB-64, American Type Culture Collection; Manassas, VA), was maintained in complete medium.
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