Whole blood from healthy females over the age of 18 years was purchased from Innovative Research (Novi, MI). Monocytes were enriched by negative selection using the Rosette Sep® monocyte enrichment cocktail according to manufacturer’s instructions (STEMCELL Technologies; Vancouver, Canada). To differentiate isolated monocytes into the AAM phenotype, monocytes were seeded on 9 mm square coverslips at a density of 200,000 cells/well for 6 days in AIM V media supplemented with 1% penicillin-streptomycin and 20 ng/mL M-CSF. Macrophages were polarized for 48 hours with 2 ng/mL IL-4 and 2 ng/mL IL-13. AAMs were washed with phosphate buffered saline (PBS) and changed to 1:1 Medium199:MCDB105 supplemented with 1% penicillin/streptomycin (serum free media, SFM) for 24 hours. Control conditions (-AAMs) were prepared by exposing cell-free coverslips to the differentiation protocol to account for the effects of non-specific adsorption of differentiation factors.
Rosette sep monocyte enrichment cocktail
Manufactured by STEMCELL
Sourced in Canada
The Rosette Sep® monocyte enrichment cocktail is a laboratory product designed to isolate monocytes from biological samples. It contains reagents that facilitate the removal of unwanted cell types, allowing for the enrichment of monocytes. The core function of this product is to provide a tool for researchers to obtain purified monocyte populations for further analysis and experimentation.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Zen 2, by Zeiss (1 mentions)
Alex fluor 488 secondary, by Thermo Fisher Scientific (1 mentions)
Aim 5 media, by Thermo Fisher Scientific (1 mentions)
Axiocam 506 mono camera, by Zeiss (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Axio observer z1 inverted microscope, by Zeiss (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Navios, by Beckman Coulter (1 mentions)
Mem non essential amino acids solution, by Fujifilm (1 mentions)
96 well flat bottom plate, by BD (1 mentions)
Penicillin streptomycin solution, by Fujifilm (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
6 protocols using rosette sep monocyte enrichment cocktail
1
Differentiation of Monocytes into Alternatively Activated Macrophages
2
Monocyte Enrichment and RNA Isolation Protocol
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For enrichment of monocytes and RNA isolation, the same methodological approach was used at both time points [18 (link)]. In brief, for monocytes enrichment the RosetteSep Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, Canada) was used directly after blood sampling. Total RNA was extracted on the same day of blood sampling using Trizol extraction (Invitrogen/Thermo Fisher, Darmstadt, Germany) and purification by the RNeasy Mini Kit (Qiagen, Hilden, Germany). Monocyte enrichment and RNA isolation were performed in the GHS study center by the same personnel for both baseline (BL) and follow up (FU) visit. The integrity of the total RNA was assessed through analysis on an Agilent Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany). Samples with a RNA integrity number (RIN) <7 were excluded. Total RNA was stored at –80°C until further processing; Time of storage was 2–14 month at both time points with the same time span for RNA samples of the same individuals at BL and FU.
3
Monocyte-Derived M2 Macrophage Generation
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Whole blood from healthy females over the age of 18 years was purchased from Innovative Research (Novi, MI). Monocytes were enriched by negative selection using the Rosette Sep® monocyte enrichment cocktail according to manufacturer's instructions (STEMCELL Technologies; Vancouver, Canada). To differentiate isolated monocytes into the M2 phenotype, 9 mm square coverslips were placed in individual wells of a 24 well plate and monocytes were seeded at a density of 200,000 cells/well for 6 days in AIM V media (Invitrogen) supplemented with 1% penicillin-streptomycin in the presence of 20 ng/mL M-CSF (Peprotech; Rocky Hill, NJ). Macrophages were then activated for 48 hours in 2 ng/mL each of IL-4 and IL-13 (Peprotech). Phenotypical characterization of MDM was performed by immunofluorescence after 8 days using anti-CD68 (clone Y1/82A), anti-CD206 (clone 19.2), and their respective isotypes (BD Biosciences; Franklin Lakes, NJ) with a goat anti-mouse Alex Fluor 488 secondary (Invitrogen). Fixed cells were imaged at room temperature in phosphate buffered solution (PBS, Invitrogen) on a Zeiss Axio Observer.Z1 inverted microscope with an AxioCam 506 mono camera, a Plan-NEOFLUOR 20x 0.4-NA air objective, and Zen2 software (Zeiss; Oberkochen, Germany).
4
Monocytic RNA Isolation and DNA Extraction
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Monocytic RNA was isolated as described previously [24 (link)]. Blood was collected using the Vacutainer CPT Cell Preparation Tube System (BD Biosciences, San Jose, CA, USA) and monocytes were enriched by negative selection with the Rosette Sep Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, BC, Canada), which leads to 72% to 85% of enriched monocytes. Total RNA was isolated using Trizol extraction and purification by silica-based columns. Genomic DNA from leukocytes was extracted as described by Miller et al. from buffy-coated ethylenediamine tetraacetic acid blood samples [24 (link),25 (link)].
5
Isolation and Characterization of Human Peripheral Monocytes
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Peripheral blood samples were obtained from healthy volunteers after their informed consent and approval by the Ethics Committee, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove (reference number 201902 S22P), Czech Republic. Peripheral monocytes were isolated from whole blood according to the manufacturer’s instructions. Following incubation with 50 μL/mL of RosetteSep™ Monocyte Enrichment Cocktail (STEMCELL Technologies Inc., Vancouver, Canada), gradient density centrifugation (1200× g, 20 min, RT) was performed using Histopaque®-1077 (Sigma-Aldrich, St. Luis, MO, USA). The purity (~85%, Figure S2 ) of isolated monocytes was evaluated by flow cytometry (Navios™, Beckman Coulter, Brea, CA, USA). A more detailed description of their isolation and phenotyping is provided in the Supplementary Materials .
6
Monocyte Immune Response Profiling
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Peripheral blood from HC was collected in BD Vacutainer™ Glass ACD Solution A tube (Becton Dickson). Monocytes were purified using Roset-teSep™ Monocyte Enrichment Cocktail (STEMCELL Technologies) in accordance with the manufacturer's instruction. The purity of monocytes was confirmed to be >85% by flow cytometry according to the expression of CD14. Purified monocytes were suspended in RPMI 1640 (Thermo Fisher Scientific) with 10% of heat-inactivated (56℃, 20 minutes) autologous serum, MEM Non-Essential Amino Acids Solution (FUJIFILM Wako Pure Chemical Corporation), 50 nM of 2-Mercaptoethanol (Sigma Aldrich) and Penicillin-Strepto-mycin Solution (FUJIFILM Wako Pure Chemical Corporation), and were seeded on 96-well flat-bottom plate (Falcon) at a concentration of 2 x 10 5 cells/well. Then, the cells were incubated with 5% serum from a healthy donor or an SLE patient for 16 hours to evaluate FLICA™-caspase-1, or for 24 hours to analyse with enzyme-linked immunosorbent assay (ELISA). In some experiments, monocytes were pre-treated with small chemical inhibitors for one hour prior to incubation with serum.
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