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.
Enzyme d
Manufactured by Miltenyi Biotec
Sourced in Germany
Enzyme D is a laboratory reagent used for the digestion of biological samples. It is a highly purified enzyme that catalyzes the breakdown of specific molecular bonds within the sample, preparing it for further analysis or processing. The core function of Enzyme D is to facilitate the preparation of samples for downstream applications in biotechnology and life science research.
Automatically generated - may contain errors
Lab products found in correlation
Enzyme a, by Miltenyi Biotec (2 mentions)
Cell lysing solution, by BD (1 mentions)
Fitc labeled anti cd11b, by Thermo Fisher Scientific (1 mentions)
Anti cd16 32 clone 2.4g2, by BD (1 mentions)
100 μm cell strainer, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Gentlemacs octo dissociator with heater, by Miltenyi Biotec (1 mentions)
Antibiotic antimycotic abam, by Thermo Fisher Scientific (1 mentions)
2 protocols using enzyme d
1
Isolation and Analysis of Lung Immune Cells
2
Isolation of Vascular Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The resected tissue specimens were placed in Dulbecco’s modified Eagle’s medium (DMEM; Fujifilm Wako, Osaka, Japan) supplemented with Antibiotic-Antimycotic (ABAM; Thermo Fisher Scientific, Waltham, MA, United States). The specimens were transported to the laboratory and cut into 1 × 2-cm with full thickness. The VECs were then isolated by washing the tissues three times with DMEM supplemented with ABAM, mincing the tissues into approximately 3-mm3 pieces with scissors, and digesting the mix with Skin Dissociation Kit (Miltenyi Biotech, Bergish Gladbach, Germany) according to the manufacturer’s instructions. Briefly, this process involved placing the tissue mix in a gentleMACS C tube together with DMEM containing 1% bovine serum albumin (BSA), Enzyme P, Enzyme D, and Enzyme A (Miltenyi Biotech). The mixture was then allowed to digest for approximately 1 hour in a GentleMACS Octo Dissociator with Heaters (Miltenyi Biotech). Subsequently, the cells were squeezed through 100-μm Cell Strainers (Corning, Corning, NY, United States) and harvested by centrifugation (300 g, 10 min, 4°C). Thereafter, PEB buffer composed of phosphate-buffered saline (PBS), pH 7.2, 0.5% BSA, and 2 mM EDTA was added to the cells and mixed well.
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