After anesthesia, the lungs were flushed in situ with 5 mL PBS through heart cannulation to remove the intravascular blood pool. Lung tissues were minced and digested with 200 μg/mL collagenase D and 40 μg/mL DNase I (both from Roche) in 10 mL RPMI 1640 medium at 37 °C for 1 h on a shaker. Subsequently, the enzymatically digested lung tissues were filtered through a stainless steel mesh. Annexin V microbeads were used to remove apoptotic cells, following the manufacturer’s instructions (Miltenyi Biotech, Bergisch Galdbach, Germany). Viable neutrophils were then prepared using the neutrophil isolation kit (Miltenyi Biotech, Bergisch Galdbach, Germany) and resuspended in RPMI 1640 medium containing 10% fetal calf serum and antibiotics.
Annexin 5 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
Annexin V microbeads are magnetic beads coated with the protein Annexin V. Annexin V has a high affinity for phosphatidylserine, a phospholipid that is exposed on the surface of apoptotic cells. These microbeads can be used to detect and isolate apoptotic cells from a cell population.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (1 mentions)
Collagenase d, by Roche (1 mentions)
Neutrophil isolation kit, by Miltenyi Biotec (1 mentions)
Mini tube rotator, by Thermo Fisher Scientific (1 mentions)
Macs streptavidin microbeads, by Miltenyi Biotec (1 mentions)
Sodium orthovandate, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hema 3, by Thermo Fisher Scientific (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Anti ly6g microbead kit, by Miltenyi Biotec (1 mentions)
Minimacs, by Miltenyi Biotec (1 mentions)
5 protocols using annexin 5 microbeads
1
Isolation of Viable Lung Neutrophils
2
Quantifying SARS-CoV-2 Viral Load in Cells
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1.0 × 106 isolated CoV2-AC and their equivalent purified conditioned supernatants, obtained as described above, were lysed by snap freeze-and-thaw. To estimate viral load in AC isolated by annexin V labeling, all infected cells were collected, followed by magnetic separation with annexin V microbeads (Miltenyi Biotec) according to the manufacturer’s protocol. 1.0 × 106 pre-sorted cells and annexin+ cells were lysed by freeze-and-thaw for quantification. Viral loads were estimated by titration in Vero CCL81 cells seeded onto 96-well plates and expressed as 50% tissue culture infectious dose (TCID50). Quantification was performed with the Reed-Muench method and plotted as TCID50 units per mL (Reed and Muench, 1938 (link)).
3
Isolation and Analysis of Extracellular Vesicles
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Platelet‐free plasma samples (from 18 mL ACD‐A anticoagulated blood/person) of three HSs were pelleted at 20,000 g, 40 min, 16°C, after which the supernatants were removed carefully with a needle and syringe. The pellets were then re‐suspended in 300 μL Annexin‐binding buffer (ABB) (BD Pharmingen). Meanwhile, 10 μL of Streptavidin MicroBeads (MACS, Miltenyi Biotec) was incubated with 10 μL Annexin V MicroBeads (MACS, Miltenyi Biotec) per sample for 30 min at RT, with low‐speed rotation (Fisherbrand Mini Tube Rotator, 10 rpm). Beads were then transferred into the blood plasma mEV samples (20 μL of the mixed beads into each sample) and were incubated for 60 min at RT, with low‐speed rotation. Samples were then loaded onto μ Columns (MACS, Miltenyi Biotec) that were previously equilibrated with 0.1% Triton X‐100 and washed four times with ABB. Columns were then washed six times with ABB. Samples were eluted with 100 μL protein lysis buffer (Raybiotech Inc.). Protease inhibitors were added to the samples (protease inhibitor cocktail (Sigma), 0.005 M phenylmethylsulphonyl fluoride (PMSF, Sigma), 0.001 M sodium orthovandate (Sigma)), after which samples were frozen‐thawed three times and sonicated for 20 min. Protein concentration was determined with a Micro BCA assay. Samples were then kept frozen at ‐20°C till they were used for Wes analysis.
4
Isolation of BM-derived Neutrophils
5
Sperm Apoptosis Detection Protocol
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The sample was incubated with 100 μl 1×BB and annexin-V microbeads (Miltenyi Biotec SAS) for 15 min at room temperature. An additional aliquot was kept for the study of DNA fragmentation, and the remaining spermatozoa-microbead mix was placed on the separation column containing magnetic beads (MiniMACS, Miltenyi Biotec SAS). The spermatozoa negative for EPS passed through the column and were considered as annexin V (−) fraction. Elution using 1×BB allowed the collection of annexin V (+) spermatozoa, corresponding to sample with EPS. The externalization of phosphatidylserine, DNA fragmentation and aneuploidy rate were carried out on both annexin V (−) and annexin V (+) fractions of each patient.
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