Bone marrow (BM) cells were cultured 7 days in IMDM or RPMI 1640 supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin (Invitrogen, San Diego, CA), 10% heat inactivated fetal bovine serum (Biochrom GmbH, Berlin, DE) and 50 U/ml macrophage colony-stimulating factor (ImmunoTools, Friesoythe, Germany) or 30% L929 cell supernatant to generate BM-derived macrophages (BMDMs) (43 (link), 44 (link)). Cells were seeded in half-area 96-well plates (2.5 × 104 cells/well), 96-well plates (2 × 105 cells/well) and 6-well plates (3 × 106 cells/well) without growth factors. Neutrophils were isolated from the bone marrow using the Neutrophil isolation kit (Miltenyi, Bergisch Gladbach, Germany) and plated in 96-well plates (105 cells/well). Salmonella minnesota ultra pure lipopolysaccharide (LPS) was from List Biologicals Laboratories (Campbell, CA), Pam3CSK4 from EMC microcollections GmBH (Tübingen, Germany), and CpG ODN 1826 (CpG) and poly(I:C) from Invivogen (San Diego, CA). Monosodium urate (MSU) crystals were prepared as described (45 (link)). Listeria monocytogenes 10403 s was grown in brain heart infusion broth (BD Biosciences, Erembodegem, Belgium). Bacteria were washed with 0.9% NaCl and adjusted at 1010 cfu/ml. When required, bacteria were heat-inactivated for 2 h at 70°C.
Neutrophil isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The Neutrophil Isolation Kit is a laboratory product designed to isolate neutrophils from whole blood samples. It utilizes a proprietary method to separate and enrich the neutrophil population for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by BioLegend (2 mentions)
Thp1 cells no tib 202, by American Type Culture Collection (2 mentions)
Lineage cell depletion kit, by Miltenyi Biotec (2 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions)
Monocyte isolation kit, by Miltenyi Biotec (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Macrophage colony stimulating factor, by Immunotools (1 mentions)
Brain heart infusion broth, by BD (1 mentions)
Zymosan, by Merck Group (1 mentions)
C57bl 6 mice, by Charles River Laboratories (1 mentions)
Monocyte nucleofector kit, by Lonza (1 mentions)
Lipofectamine rna interference max transfection reagent, by Thermo Fisher Scientific (1 mentions)
Neutrophil elastase activity assay kit, by Cayman Chemical (1 mentions)
Rt pcr kit, by Takara Bio (1 mentions)
Il 10, by BioLegend (1 mentions)
Pe anti cd11b antibodies, by BioLegend (1 mentions)
Tgf β elisa kits, by BioLegend (1 mentions)
90 protocols using neutrophil isolation kit
1
Generation of Bone Marrow-Derived Macrophages
2
Isolation of Human and Mouse Neutrophils
3
Isolation and characterization of mouse neutrophils
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C57BL/6 mice were obtained from Charles River UK. PSD-95−/− mice were obtained from the Wellcome Trust Sanger Institute. Female and male mice aged 7–12 weeks were used, with each serving as their own control. Neutrophils were isolated from either bone marrow or peritoneal exudates. Bone marrow derived neutrophils, which have similar respiratory burst and phagocytic characteristics to blood neutrophils [22 (link)], were extracted from age and gender-matched mice after cervical dislocation to avoid the potential confounding effects of inhalational anaesthesia on NMDAR activity [10 (link)]. Non-viable cells were excluded by 7AAD staining. Neutrophils were identified by Ly6G+Ly6C−CD11bhi staining. For peritoneal neutrophil harvest, zymosan (1 mg·g−1; Sigma, UK) was injected intraperitoneally in C57B/6 wild-type mice of either gender (age 6–8 weeks) prior to lavage with 5 ml ice cold PBS 3 h later. Highly purified neutrophils were obtained by negative selection using a neutrophil isolation kit (MACS Miltenyi Biotec), achieving ≥98% purity.
4
Isolation and Transfection of Murine Neutrophils
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Neutrophils in BALF and bone marrow (BM) from mice were isolated using Neutrophil Isolation Kit (Miltenyi Biotec) according to manufacturer’s guideline. To obtain BALF, mice were sacrificed by over dosage avertin. The trachea was exposed using scissors. Using a 23 G needle to slowly inject 1 ml cold PBS with 0.1 mM EDTA into the lung, then collect the BALF from lungs. To obtain BM, femurs and shins were isolated with scissors from sacrificed mice, and then the bits of bone were removed. Flush the femurs and shins with 3 ml cold RPMI-1640 medium using a 25 G needle. The BM neutrophils were cultured in RPMI-1640 medium supplemented with 10% FBS and 1000 U/ml penicillin and 1000 μg/ml streptomycin. For plasmid transfection, the expression vector was transfected into BM neutrophils using the monocyte nucleofector kit (Lonza) according to the manufacturer’s instructions. Delivery of HMGB1-siRNAs (RIBOBIO) or PAFR-siRNAs (Santa Cruz) into cells was carried out with Lipofectamine RNA interference MAX Transfection Reagent (Life Technologies) following the manufacturer’s instructions.
5
Neutrophil Elastase Activity Assay
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Neutrophils were purified from WT and KI bone marrow by negative selection using a Neutrophil Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Purified neutrophils were suspended in 1% BSA/RPMI 1640, seeded onto a 96-well plate (1 x 106 cells/well), and activated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 4 h. Elastase activity was detected from cell supernatants with a Neutrophil Elastase Activity Assay Kit (Cayman Chemicals), according to the manufacturer’s instructions.
6
Neutrophil Activation by TLR2 Agonist
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TLR2 agonist Pam3CSK4 was synthesized from InvivoGen (San Diego, USA). PE/CY5.5-anti- Gr-1, PE-anti-CD11b antibodies, TNF-α, IL-6, IL-10, IL-1β and TGF-β ELISA kits were purchased from BioLegend Inc. (San Diego, USA); PE/CY5.5 Rat IgG2b (κ) and PE Rat IgG2b (κ) were used as isotype controls (BioLegend Inc, San Diego, USA). methicillin-resistant S. aureus (ATCC43300) was purchased from Wenzhou Kont Biology & Technology Co. LTD (Jiangsu, China); Fluorescein isothiocyanate (FITC) was purchased from Sigma (Saint Louis, USA); Neutrophil Isolation Kit was purchased from Miltenyi Biotec (Cologne, Germany); TRIzol solution, a reverse transcription-PCR (RT-PCR) Kit and fluorescence quantitative PCR Kit were purchased from Takara Bio Inc. (Tokyo, Japan). Primers were synthesized by Invitrogen Inc. (Shanghai, China). Neutrophils Oxidative Burst Quantitative Assay Kit and ELISA Kit for Lactoferrin were purchased from Absin Inc. (Shanghai, China) and Cloud-Clone Corp. (Houston, USA), respectively.
7
Neutrophil and T-cell Aging Protocol
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Neutrophils were isolated from bone marrow cell suspensions by negative selection using the Neutrophil Isolation Kit (Miltenyi) following the manufacturer’s instructions. aNs were generated by aging for 24 hours in complete media containing 90% RPMI 1640, 5% FBS, 0.5% gentamicin, and 1% l -glutamine. Thymocytes were isolated via disruption of thymic tissue. aTs were generated by aging overnight in complete media containing 90% RPMI 1640, 10% FBS, 0.5% gentamicin, and 1% l -glutamine. Apoptosis was verified by annexin V and PI staining. aTs were then labeled with eF450 dye following the manufacturer’s instructions and used for coculture experiments.
8
Isolation of Viable Lung Neutrophils
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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.
9
HVJ-E Induces Neutrophil Conversion
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Example 13
Whether HVJ-E directly acts on neutrophil to induce the conversion of neutrophil to N1 type neutrophil was examined. Neutrophil was separated by MACS with Neutrophil Isolation Kit (Miltenyi Biotec serial number 130-097-658) according to the use explanation of the Kit from the cells separated from the mouse bone marrow. After 2×105 neutrophils were seeded in a 96 well plate, HVJ-E was added at 500 m.o.i. and the expression of Fas and ICAM-1, which are N1 type neutrophil markers, was analyzed by flow cytometry (FACS) 24 hr later. As a result, the neutrophil cocultured with HVJ-E was converted to N1 type neutrophil (
10
Establishing MHV-3 and APAP-Induced Liver Failure Models in Mice
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A total of 100 plaque forming unit (PFU) of MHV-3 was injected intraperitoneally into the mice to establish a FVH model. MHV-3 was obtained from the American Type Culture Collection (Manassas, VA) and expanded according to a previously published protocol.14 (link) APAP (500 mg/kg) was used to establish a mouse model of non–virus-caused ALF. NETs were depleted by intraperitoneal injection of DNase I (750 U), a widely used method to remove the DNA scaffold of NETs,8 (link) at 4 hours and 48 hours after MHV-3 infection. For neutrophil depletion, mice were injected intraperitoneally with anti-Ly6G (clone 1A8; BioXcell, West Lebanon, NH) and rat IgG2a (clone 2A3; BioXcell, West Lebanon, NH) were used as an isotype control. To adoptively transfer neutrophils, BM neutrophils were purified using the Neutrophil Isolation Kit (Miltenyi Biotec, San Diego, CA). A total of 4 × 106 WT or fgl2-/- neutrophils were adoptively transferred into MHV-3–infected fgl2-/- mice by intravenous injection.
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