At sacrifice, the right lung was dissected into single lobes and then digested with an enzyme mixture (buffer S, enzyme D, and enzyme A from Miltenyi Biotec, Bergisch Gladbach, Germany). After filtration and centrifugation, the resulting cell suspensions were filtered through a 70-μm nylon mesh, and red blood cells were lysed with a cell lysing solution (BD Biosciences Pharmingen, San Diego, CA, USA). Then, the cells were incubated in saturating doses of purified anti-mouse Fc receptor (anti-CD16/32, clone 2.4G2, BD Bioscience) in 1 mL of PBS + 5% FCS for 10 min on ice to prevent antibody binding to the Fc receptor. The following antibodies were used for surface staining (eBioscience): PE-labeled anti-CD45, FITC-labeled anti-CD11b, HorizonTM BV480-labeled anti-CD11c, PerCP-Cy5.5-labeled anti-Ly6C and PE-Cy7-labeled anti-Ly6G. A PE annexin V apoptosis detection kit was used to detect apoptosis in RAW264.7 cells according to the manufacturer’s protocol.
Cell lysing solution
Manufactured by BD
Sourced in United States
The Cell Lysing Solution is a reagent designed to disrupt and lyse cells, releasing their intracellular contents. It is a key tool for sample preparation in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Enzyme a, by Miltenyi Biotec (1 mentions)
Enzyme d, by Miltenyi Biotec (1 mentions)
Fitc labeled anti cd11b, by Thermo Fisher Scientific (1 mentions)
Anti cd16 32 clone 2.4g2, by BD (1 mentions)
Cd11c fitc, by BD (1 mentions)
Cd123 pe, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
Cd11c pe, by BD (1 mentions)
2 protocols using cell lysing solution
1
Isolation and Analysis of Lung Immune Cells
2
Quantifying Dendritic Cell Subsets
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Twenty ml of heparinized peripheral blood was obtained from the controls and lung cancer patients. The following monoclonal antibodies were added to the fresh blood, which was cultured at room temperature for 2 h: Lineage-1, fluorescein isothiocyanate (FITC; BD Biosciences, San Jose, CA, USA); HLA-DR, Per-CP (BD Biosciences); CD11c, FITC (BD Biosciences); CD11c, PE (BD Biosciences); and CD123, PE (BD Biosciences). Thereafter, erythrocytes were hemolyzed by cell lysing solution (BD Biosciences). The DC subtype and percentage of each subtype were analyzed by flow cytometry (FACSCalibur, BD Biosciences). Data were analyzed using CellQuest Pro (BD Biosciences). After gating mononuclear cells based on side scatter and forward scatter, the blood DC population was identified as the lin−/HLA-DR+ fraction. DCs were divided into a CD11c+DC subset (mDCs) and a CD123+DC subset (pDCs). The number of total events was 200,000, and data are expressed as DC counts per 200,000 leukocytes.
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