Human peripheral blood sample from health volunteers or patients with CAD were obtained from the Ningbo First Hospital, which was allowed by the ethics committee of Ningbo First Hospital. Human primary macrophages were induced from monocytes, which were separated from human peripheral blood with human lymphocyte separation medium (Sigma).
Human lymphocyte separation medium
Manufactured by Merck Group
Human lymphocyte separation medium is a laboratory reagent used to isolate and purify human lymphocytes from whole blood samples. It is designed to facilitate the separation of lymphocytes from other blood components through density gradient centrifugation.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Il 1β elisa kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Rpmi 1640 culture medium, by GE Healthcare (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Fitc labeled anti cd3 antibody, by BD (1 mentions)
Rt qpcr kit, by Qiagen (1 mentions)
Anti nlrp3, by Cell Signaling Technology (1 mentions)
Anti caspase 1, by Cell Signaling Technology (1 mentions)
Anti il 1β, by Cell Signaling Technology (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Recombinant human gm csf, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ox ldl, by Beyotime (1 mentions)
Ifn γ, by BioLegend (1 mentions)
3 protocols using human lymphocyte separation medium
1
Isolation of Human Primary Macrophages
2
Immunological Characterization of NLRP3 Inflammasome
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Human lymphocyte separation medium, 4-aminopyridine (4-AP), N-2-hydroxyethylpiperazine-N'-2′-ethanesulfonic acid (HEPES), and K+ asparate were obtained from Sigma-Aldrich (St. Louis, MO, USA). The TRIzol reagent, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) solution and the reverse transcription kit (catalog no. K1622) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) kit (catalog no. 204054) was obtained from Qiagen (Hilden, Germany). In addition, the anti-NLRP3 (catalog no. 13158S; 1:1,000), anti-caspase-1 (catalog no. 3866S; 1:1,000), anti-IL-1β (catalog no. 31202S; 1:1,000) and anti-GAPDH (catalog no. 2118S; 1:1,000) rabbit monoclonal antibodies, as well as the anti-rabbit IgG, alkaline phosphatase-linked secondary antibody (catalog no. 7054S, 1:2,000), were from Cell Signaling Technology, Inc. (Beverly, MA, USA). The IL-1β ELISA kit (catalog no. BMS224/2) was obtained from eBioscience (San Diego, CA, USA), while the FITC-labeled anti-CD3 antibody (catalog no. 340571) and recombinant human IL-2 (rIL-2) were from BD Biosciences (San Jose, CA, USA). The RPMI 1640 culture medium and fetal bovine serum were from GE Healthcare (Chicago, IL, USA). The other patch-clamp associated reagents were obtained from Amresco (Solon, OH, USA).
3
Endothelial and Macrophage Cell Culture Protocols
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Human umbilical vein endothelial cells (HUVECs), and human monocyte-like cell line (THP-1) were cultured in HUVEC specific medium (PROCELL, CHINA, Ham's F-12 K, 0.1 mg/mL Heparin, 0.03–0.05 mg/mL ECGs, 1% P/S) or RPMI-1640 (Gibco, USA) medium supplied with 10% fetal bovine serum (Gibco, USA). The THP-1 cells were stimulated with phorbol ester (PMA) (100 nM, sigma, USA) to differentiate macrophage-like sticky cells for later experiments. Peritoneal macrophages were isolated as previously described [14 ]. Human peripheral blood sample from healthy volunteers (HV) or patients with coronary artery disease (CAD) were obtained from the Ningbo First Hospital, which was allowed by the ethics committee of Ningbo First Hospital. Human peripheral blood-derived mononuclear cells, separated from human peripheral blood with human lymphocyte separation medium (Sigma), were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and then were differentiated into macrophages in the presence of 50 ng/ml of recombinant human GM-CSF (Peprotech) for 4 days, as previously described [17 –19 (link)]. IFN-γ was purchased from BioLegend. ox-LDL and recombinant human TNF-α protein were purchased from Beyotime.
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