CC were prepared as described elsewhere (Samstad et al. 2014 (link)). Briefly, 100 mg of ultrapure cholesterol (Sigma Aldrich) was dissolved in 50 ml of 1-propanol. The solution was mixed with sterile water (1:1.5) and allowed to rest for 20 min for the crystals to stabilize. All the steps were performed in sterile condition and at room temperature. 1-Propanol was removed by evaporation and CC were resuspended in PBS/0.05% human serum albumin (HSA). CC were further tested for endotoxin contamination using Limulus amebocyte lysate assay (LAL) and LPS levels were found to be below the detection limit (0.01EU/ml). The prepared CC were of size range 1–2 μm and stored at 4 °C.
Ultrapure cholesterol
Manufactured by Merck Group
Ultrapure cholesterol is a high-purity laboratory product used for various research and analytical applications. It provides a standardized source of cholesterol for experiments and analyses that require a consistent and well-defined cholesterol compound.
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Lab products found in correlation
Perfecta qpcr fastmix, by Quanta Biosciences (1 mentions)
Anti cd15, by BD (1 mentions)
Recombinant c5a, by Merck Group (1 mentions)
1 propanol, by Merck Group (1 mentions)
Anti cd20, by Agilent Technologies (1 mentions)
Tumor necrosis factor (tnf), by Genentech (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Anti cd3, by Agilent Technologies (1 mentions)
Anti cd68, by Agilent Technologies (1 mentions)
2 protocols using ultrapure cholesterol
1
Preparation of Cholesterol Crystals
2
Immune Profiling of Lepirudin-treated PBMCs
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PBMC were isolated from whole-blood anticoagulated with lepirudin (Refludan®, Celgene). Recombinant C5a, ultrapure cholesterol, 1-propanol were purchased from Sigma-Aldrich, and TNF from Genentech. The following reagents were used for qRT-PCR analyses: RNeasy Mini kit (Qiagen), DNase and High Capacity RNA-to-cDNA Kit (Applied Biosystems) and PerfeCTa® qPCR FastMix™ (Quanta Biosciences). Assays were from Applied Biosystems: GAPDH (Hs99999905_m1), NLRP3 (Hs00918082_m1), TNF (Hs00174128_m1), and IL-1β (Hs01555410_m1) and primers from SYBR as shown in Supplementary Table I. Antibodies used for immunohistochemistry: polyclonal rabbit anti-human C1q (A0136, Dako), normal rabbit IgG (Ab105-C, R&D systems), goat anti-mouse conjugated to Alexa 647 (Invitrogen), anti-C5b-9 (DIA-011, Antibodyshop), anti-CD68 (Dako Agilent M0814), anti-CD20 (Dako Agilent M0755), anti-CD3 (Dako Agilent A0452), anti-CD15 (BD Pharmingen 559045), control IgG2a (BD Pharmigen). Antibodies used for flow cytometry included anti-CD14 FITC (M5E2), anti-CD4 PerCP-CY 5.5 (RPA-T4), anti-CD8 BV605 (SK1) and anti-CD19 BV711 (SJ25C1) and were purchased from BD Pharmigen, and anti-CD3 APC (UCHT1) were from Invitrogen. Fluorescence-minus-one (FMO) was used as control.
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