For the isolation of the whole neutrophil population, the EasySepTM Direct Human Neutrophil Isolation Kit (STEMCELL Technologies, Canada) was used. Neutrophils were isolated according to the manufacturer’s instructions with slight modifications to increase cell yield and purity. In the first step, 50 μl of 0.5 M EDTA/1 ml blood was added, and the first incubation was extended to 6 min. Isolated cells were washed once with PBS and counted. Cell viability and purity were assessed by Trypan blue and flow cytometry, respectively. The purity of the isolated neutrophil population was higher than >96%. Mononuclear cell fraction was obtained as follows. The whole cord or peripheral blood was diluted 1:1 with PBS and layered carefully over Ficoll-PaqueTM PLUS (density 1.077 g/l) in a conical tube. Density-gradient centrifugation was performed at 500 x g for 30 min. After centrifugation, blood was separated into CBMC or PBMC-rich layer and granulocyte-rich erythrocyte pellet. Cells from both fractions were washed twice with PBS and counted. LDN were isolated from CBMC using EasySepTM Human CD15 Positive Selection Kit (STEMCELL Technologies, USA) according to the manufacturer’s instructions. HDN were isolated using EasySepTM Direct Human Neutrophil Isolation Kit (STEMCELL Technologies, USA) from the erythrocyte fraction as described above.
Easysep direct human neutrophil isolation kit
Manufactured by STEMCELL
Sourced in Canada, United States, United Kingdom, France
The EasySep Direct Human Neutrophil Isolation Kit is a magnetic cell separation system designed to isolate human neutrophils from whole blood or bone marrow samples. The kit utilizes an immunomagnetic labeling approach to selectively target and separate neutrophils from the sample.
Automatically generated - may contain errors
Lab products found in correlation
Easysep mouse neutrophil enrichment kit, by STEMCELL (6 mentions)
Sytox green, by Thermo Fisher Scientific (6 mentions)
Easysep magnet, by STEMCELL (4 mentions)
Aim 5 medium, by Thermo Fisher Scientific (3 mentions)
Lps from e coli o55 b5, by Merck Group (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Mouse neutrophil isolation kit, by Miltenyi Biotec (2 mentions)
Midimacs separator, by Miltenyi Biotec (2 mentions)
Dml28, by Caisson (2 mentions)
Fetalplex animal serum complex, by Caisson (2 mentions)
Fetalplex animalserum complex, by Gemini Bio (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
1300exi cytometer, by Stratedigm (2 mentions)
Pancoll separating solution, by PAN Biotech (2 mentions)
Pharm lyse buffer, by BD (2 mentions)
Pancoll, by PAN Biotech (2 mentions)
Edta coated vacutainer, by BD (2 mentions)
Luna fl dual fluorescence cell counter, by Logos Biosystems (2 mentions)
Edta coated vacutainer tubes, by Greiner (2 mentions)
128 protocols using easysep direct human neutrophil isolation kit
1
Isolation and Purification of Neutrophils
2
Isolation and Culture of Human and Mouse Neutrophils
3
Isolation of Human Neutrophils
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EDTA-anticoagulated peripheral blood from healthy donors was used to isolate neutrophils using EasySep Direct Human Neutrophil Isolation Kit (StemCell Technologies, USA) following the manufacturer’s instructions. Erythrocytes were lysed with red blood cell lysis buffer and washed with PBS. Subsequently, purity of the human neutrophils was evaluated by FACS using antibodies against Ly-6G/Ly-6C (1:500 dilution, Ly-6G/Ly-6C monoclonal antibody, PE, Thermo, USA) and CD11b (1:500 dilution, CD11b monoclonal antibody, FITC, Thermo, USA). Neutrophil purity was >93% (Supplementary Fig. 4 ).
4
Neutrophil Extracellular Trap (NET) Induction
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Neutrophils were isolated from freshly collected whole blood of healthy adults or adult mice using the EasySep Direct Human Neutrophil Isolation kit (19666, Stemcell Technologies) or EasySep Mouse Neutrophil Enrichment Kit (19762, Stemcell Technologies), respectively, with greater than 95% purity (63 (link)). Neutrophils were resuspended to a concentration of 1 × 106 cells/mL in Medium 199. Platelets were purified as described previously (66 (link), 67 (link)), resuspended to 1 × 108 cells/mL in Medium 199, and activated with 50 ng/mL convulxin (sc-202554, Santa Cruz Biotechnology) for 15 minutes. Activated platelets and neutrophils were incubated at a 100:1 ratio to induce NETs for 2.5 hours at 37°C in 5% CO2/95% air. HMGB1 was blocked by 10 μg/mL BoxA (HM-014, HMGBiotech). Neutrophils were pretreated for 1 hour with either nNIF or SCR peptides. NET levels were measured using the aforementioned MPO-DNA ELISA.
5
Neutrophil Isolation and Stimulation
6
Neutrophil Isolation and Migration
7
Neutrophil Isolation and Elastase Assay
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Neutrophils were isolated from human peripheral whole blood [IRB #13-3454] by immune-magnetic negative selection (EasySep™ Direct Human Neutrophil Isolation Kit, STEMCELL Technologies, Vancouver, BC, Canada, cat no. 19666) as described [21 (link)]. Freshly isolated neutrophils were re-suspended in 10 mM HEPES (pH 7.6)-buffered RPMI 1640 media. At the time of experimentation, cells were transferred to Ringer’s solution (in mM: 120 NaCl, 5.2 KCl, 1.2 MgCl2, 1.2 CaCl2.2H2O, 12 NaHCO3, 24 HEPES, 10 glucose, pH 7.4) and exposed to vehicle vs. 1% or 3% Juul-Menthol e-liquid for 2 h at 37 °C. Juul e-cigarettes were purchased from local vendors in Chapel Hill, NC, USA. Media was centrifuged, and the activated neutrophil supernatants (ANS) were frozen at −80 °C until needed. Neutrophil elastase activity in naïve media or ANS was evaluated by monitoring the cleavage of a neutrophil elastase-specific fluorogenic peptide (Suc-Ala-Ala-Ala-MCA, Peptides International, Louisville, KY, USA) using an M1000 multi-plate reader (Tecan, Raleigh, NC, USA) as described [21 (link)].
8
Neutrophil Isolation from Healthy Volunteers
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Blood was collected from healthy volunteers. Neutrophil isolation was performed using a previously described method (43 (link)). Briefly, blood was collected on a Li-heparin 6-ml Vacutainer and neutrophils were isolated using negative selection with the EasySep direct human neutrophil isolation kit (Stemcell Technologies, Cambridge, United Kingdom). Once isolated, human neutrophils were suspended in RPMI 1640 without phenol red containing 1% human serum, which was also the medium in which all assays were performed. Before every experiment, neutrophils were counted and evaluated for viability using trypan blue staining and diluted to the desired concentration.
9
Neutrophil Activation and Secretome
10
Isolation of mouse and human neutrophils
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Mouse neutrophils were isolated by Mouse Neutrophil Isolation Kit (Miltenyi, #130-097-658). Briefly, 50 µL of the Neutrophil Biotin-Antibody Cocktail were added to 200 µL of a cell suspension (5×107 total cells in Magnetic-Activated Cell Sorting (MACS) buffer) and incubated for 15 min at 4°C. After washes with MACS buffer two times, 100 µL of Anti-Biotin MicroBeads were added to 400 µL of the cell suspension. An LS column and MidiMACS separator (Miltenyi) were applied for subsequent magnetic sorting.
Human PB-derived neutrophils were isolated by EasySep Direct Human Neutrophil Isolation Kit (STEMCELL, #19666). The isolation antibody cocktail (50 µL/1 mL whole blood) and RapidSpheres beads (50 µL/1 mL whole blood) were added to whole blood and incubated for 5 min at RT successively. Then, isolation buffer was added to the cell suspension to bring the volume to 10 mL, and the tube was placed in the EasySep Magnets (STEMCELL Technologies) for 5 min. The enriched cell suspension was collected in a new tube and incubated with RapidSpheres beads (50 µL/mL) for another 5 min. A second separation with EasySep Magnets for 5 min was performed, and the enriched cell suspension was collected for subsequent in vitro induction.
Human PB-derived neutrophils were isolated by EasySep Direct Human Neutrophil Isolation Kit (STEMCELL, #19666). The isolation antibody cocktail (50 µL/1 mL whole blood) and RapidSpheres beads (50 µL/1 mL whole blood) were added to whole blood and incubated for 5 min at RT successively. Then, isolation buffer was added to the cell suspension to bring the volume to 10 mL, and the tube was placed in the EasySep Magnets (STEMCELL Technologies) for 5 min. The enriched cell suspension was collected in a new tube and incubated with RapidSpheres beads (50 µL/mL) for another 5 min. A second separation with EasySep Magnets for 5 min was performed, and the enriched cell suspension was collected for subsequent in vitro induction.
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