The NETosis assay was performed as previously described [10 (link)]. Briefly, microfluidic devices were primed with RPMI media with no FBS. Neutrophils were isolated from healthy donors using the Easysep Direct Neutrophil Isolation Kit (STEMCELL Technologies). Isolated neutrophils were stained with 32 μM Hoeschst 3342 dye and mixed with SYTOX green (final concentration 2 μM). Stained neutrophils were stimulated with either pooled saliva samples or individual samples of VTM in the presence or absence of spike-coated NeutrAvidin beads. The cell suspensions were then loaded into a microfluidic device and imaged with brightfield, FITC, and DAPI fields every 10 minutes for 6 hours. NETosis was then quantified using FIJI and the TrackMate plugin.
Easysep direct neutrophil isolation kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep Direct Neutrophil Isolation Kit is a laboratory product designed for the isolation of neutrophils from whole blood samples. It utilizes a magnetic bead-based separation method to selectively isolate neutrophils from the sample.
Automatically generated - may contain errors
Lab products found in correlation
Isolation cocktail, by STEMCELL (2 mentions)
Easysep magnet, by STEMCELL (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Edta vacutainer, by BD (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Msu crystal, by InvivoGen (1 mentions)
Phorbol myristate acetate (pma), by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Penicillin streptomycin solution, by Corning (1 mentions)
Methocult h4034 optimum, by STEMCELL (1 mentions)
Easysep mouse neutrophil enrichment kit, by STEMCELL (1 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Anti flotillin 1, by BD (1 mentions)
Rapidspheres, by STEMCELL (1 mentions)
11 protocols using easysep direct neutrophil isolation kit
1
Microfluidic NETosis Assay for Neutrophil Study
2
Neutrophil Isolation from Whole Blood
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Whole blood samples were obtained from the healthy donors and anti-coagulated with K2EDTA at the Stanford Blood Center. Samples were de-identified by the Stanford Blood Center. Neutrophils were purified on the same day as isolation, and the whole blood samples were kept at room temperature between collection and neutrophil isolation. Neutrophils were isolated by EasySep Direct Neutrophil Isolation Kit (StemCell Technologies, 19666) according to the manufacturer’s instructions in 5 mL aliquots on a magnetized rack (StemCell Technologies, 18103). Efficiency of isolation was determined by flow cytometry to identify CD45 and CD14 hi cells. Neutrophils were routinely obtained in >96% purity via this method.
3
Neutrophil Isolation and Stimulation
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The human blood was collected into EDTA tubes, and neutrophils were purified using the EasySep Direct Neutrophil Isolation Kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer's protocol. Neutrophil purity was at least 95% by flow cytometry. Neutrophils were plated onto acid-washed, poly-L-lysine (Sigma Diagnostics, Livonia, USA) coated 12 mm glass coverslips at a concentration of 50,000 cells per coverslip in media containing RPMI 1640 (Thermo Fisher Scientific, Waltham, USA) with 2% fetal bovine serum (Atlanta Biologicals, Flowery Branch, USA) and 1% penicillin-streptomycin solution (Corning, Tewksbury, USA). Neutrophils were treated with the following and incubated for 4 hours at 37°C, 5% CO2: 4 μM ionomycin (MilliporeSigma, Darmstadt, Germany), 560 μg/mL MSU crystals (InvivoGen, San Diego, USA), 25 nM PMA (Fisher BioReagents, Waltham, USA), or 1 × 106Candida albicans strain SC5314 [42 (link)].
4
Neutrophil Isolation from Human Blood
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Neutrophils were isolated from fresh human peripheral blood with patient consent and approval of the Institutional Review Board (IRB) of the University of Southern California (USC), protocol #HS-20–00546. CD15-expressing neutrophils were isolated using the EasySep™ direct neutrophil isolation kit (Stem Cell Technologies, Seattle, WA) within 1 hour of the blood draw as per the manufacturer’s instructions. Briefly, 5 ml of peripheral blood was collected into 10 ml EDTA vacutainers (Becton Dickinson, Franklin Lakes, NJ). From this, 3 ml was diluted 1:1 with PBS (Thermo Fisher Scientific, Waltham, MA) and kept on ice for purity analysis by flow cytometry. The remaining 2 ml was transferred to a 5 ml polystyrene round bottomed tube (Genesee Scientific, San Diego, CA) and gently combined with 100 μl of isolation cocktail and 100 μl of RapidSpheres™(Stem Cell Technologies). After incubation at room temperature for 5 mins, 1.8 ml of 1 mM EDTA was added, gently mixed, and placed into the EasySep™ Magnet (Stem Cell Technologies) for 5 mins. The enriched cell suspension was placed into the EasySep™ Magnet for an additional 5 mins and decanted into a fresh tube. Approximately 4.25 × 106cells were isolated from 5 ml of peripheral blood.
5
Hematopoietic Progenitor Cell Differentiation
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After 14 days, dissociated EB were resuspended at a density of 4×104 cells/ml in methylcellulose (MethoCult™ H4034 Optimum, STEMCELL Technologies, Vancouver, BC, Canada) as previously described (32 (link)). After additional 12 days (day 14 + 12), hematopoietic colony-forming units (CFU) formation in the methylcellulose was assessed. Morphology of CFU was determined using an inverted light microscopy and the numbers of granulocyte CFU (CFU-G), granulocyte-monocyte CFU (CFU-GM), and granulocyte-erythroid-monocyte/macrophage CFU (CFU-GEMM), as well as erythroid CFU (CFU-E), erythroid blast-forming units (BFU-E), and monocyte CFU (CFU-M) visualized were recorded. Neutrophils were purified from CFU-G by gently dissolving the methylcellulose in PBS, followed by negative selection using the EasySep direct neutrophil isolation kit (STEMCELL Technologies, 19666, Vancouver, BC, Canada). The numbers of neutrophils were determined by hemocytometer.
6
Isolation of Murine and Human Neutrophils
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BM neutrophils were isolated from the PBS-flushed femur and tibia bones of BM of 6- to 12-week-old mice. Obtained cell suspension was washed, then resuspended at 1 × 108 cells/mL in PBS + 0.5% BSA and 2 mM EDTA. Murine neutrophils were isolated from the peripheral blood or BM using EasySep Mouse Neutrophil Enrichment Kit (STEMCELL Technologies), and human neutrophils were isolated from peripheral blood of healthy donors at Eastern Virginia Medical School using EasySep Direct Neutrophil Isolation Kit (STEMCELL Technologies).
7
Neutrophil Purification and Fixation
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Human blood samples were collected into EDTA tubes and neutrophils were purified using the EasySep™ Direct Neutrophil Isolation Kit (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. Neutrophils were determined to be 95% pure by flow cytometry. Following purification, 2.5–5 × 104 neutrophils were pipetted onto acid-washed coverslips coated with poly-L-lysine (Sigma Aldrich, St. Louis, USA) and then incubated in Roswell Park Memorial Institute (RPMI) 1640 medium for either 2 or 4 h at 37 °C, 5% CO2 followed by fixation in 70% ethanol overnight at 4 °C. Then, the neutrophils were washed with phosphate buffered saline (PBS, Corning, Corning, USA), stained with 250 nM Sytox (ThermoFisher Scientific, Waltham, USA) and washed with PBS again. Coverslips were mounted onto microscope slides with Prolong Diamond Antifade (ThermoFisher Scientific), allowed to dry, sealed with clear nail polish (Sally Hansen, New York, USA), and stored at -80 °C.
8
Neutrophil Isolation and Analysis
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Neutrophils were purified from blood samples (buffy coat, described above) by negative selection with the EasySep direct neutrophil isolation kit (19,666, STEMCELL Technologies, Vancouver, BC, Canada) according to manufacturer’s recommendations after the removal of platelet-rich plasma. See Additional file 6 and 7 for neutrophil purity (CLEC12A indicated as MICL for myeloid inhibitory C-type lectin-like receptor). Purified neutrophils were resuspended in Hanks' Balanced Salt Solution (HBSS) and lysed prior to SDS-PAGE under non-reducing (for CLEC12A) or reducing (for flotillin-1) conditions as in Gagné et al16 (link). Final antibody concentrations for immunoblotting were: 4 µg/ml anti-CLEC12A (clone 50C1, kindly provided by Dr Mireille Lahoud), 0.5 µg/ml anti-flotillin-1 (610821, BD Transduction Laboratories, Canada), and 0.05 µg/ml horseradish peroxidase-labelled donkey anti-mouse (715-035-150, Jackson ImmunoResearch Laboratories Inc., West Grove, PA,USA) antibodies. Staining was detected with the Western Lightning Chemiluminescence Plus ECL kit (PerkinElmer, Waltham, MA, USA) within a maximal exposure time of five minutes.
9
Ultrapurification of Neutrophils from Venous and Umbilical Cord Blood
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Venous and umbilical cord blood was collected by syringe into K+EDTA (Potassium EDTA) tubes (Greiner). Neutrophils were then ultrapurified using an EasySep Direct Neutrophil isolation kit (Stem Cell Technologies) according to the manufacturer’s instructions and processed as described previously.31 (link)
10
Isolation of Human Neutrophils
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Neutrophils were isolated from fresh human peripheral blood with patient consent and approval of the Institutional Review Board (IRB) of the University of Southern California (USC), protocol #HS-20-00546. CD15-expressing neutrophils were isolated using the EasySep™ direct neutrophil isolation kit (Stem Cell Technologies, Seattle, WA) within 1 hour of the blood draw as per the manufacturer’s instructions. Briefly, 5 ml of peripheral blood was collected into 10 ml EDTA vacutainers (Becton Dickinson, Franklin Lakes, NJ). From this, 3 ml was diluted 1:1 with PBS (Thermo Fisher Scientific, Waltham, MA) and kept on ice for purity analysis by flow cytometry. The remaining 2 ml was transferred to a 5 ml polystyrene round bottomed tube (Genesee Scientific, San Diego, CA) and gently combined with 100 μl of isolation cocktail and 100 μl of RapidSpheres™ (Stem Cell Technologies). After incubation at room temperature for 5 mins, 1.8 ml of 1 mM EDTA was added, gently mixed, and placed into the EasySep™ Magnet (Stem Cell Technologies) for 5 mins. The enriched cell suspension was placed into the EasySep™ Magnet for an additional 5 mins and decanted into a fresh tube. Approximately 4.25 x 106 cells were isolated from 5 ml of peripheral blood.
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