For IAV and PRV3M infections, BMDMs, BMDCs or reconstituted iMACs were first primed with 10 ng ml−1 LPS for 3 h, washed and then infected with IAV (MOI = 0.5, 1, 2, 5 or 10) for 2 h or PRV3M (MOI = 1, 2, 5 or 10) for 4 h. Subsequently, the medium was replaced with AIM V serum-free medium (Invitrogen). At 24 h post infection, cells were collected for detection of ASC specks and immunoblots, and the cell-free supernatant for IL-1β immunoblots or ELISA and virus titrations. For MERS-CoV virus infections, PBMCs were first primed with 10 ng ml−1 LPS for 3 h, washed and then infected with a series of MOIs (0.25, 0.5 or 1) for 1 h. Lower MOIs of MERS-CoV were able to elicit a robust activation of the inflammasome in human PBMCs and hence were used in this study. The medium was replaced with AIM V serum-free medium (Invitrogen) for an additional 23 h before harvesting the cells for detection of ASC specks post-fixation and the cell-free supernatant for IL-1β ELISA and virus titration. Inhibitor MCC950 (50 μM; Selleckchem) was added 1 h before the virus infection in human PBMCs.
Aim 5 serum free medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
AIM V® Serum Free Medium is a cell culture medium formulated for the growth and maintenance of a variety of cell types in the absence of animal-derived serum. It is a chemically defined, protein-free medium that supports the cultivation of various cell lines.
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Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Macrophage sfm, by Thermo Fisher Scientific (2 mentions)
X vivo15 hematopoietic cell medium x vivo15, by Lonza (2 mentions)
Immunocult xf t cell expansion medium immunocult xf, by STEMCELL (2 mentions)
Rpmi 1640 medium with l glutamine and phenol red, by Thermo Fisher Scientific (2 mentions)
Calcium and magnesium free pbs, by Merck Group (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Aim 5 serum free medium, by Merck Group (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Beckman gold 126 166nm, by Beckman Coulter (2 mentions)
Karat software, by Beckman Coulter (2 mentions)
Mcc950, by Selleck Chemicals (1 mentions)
Axiovert 25, by Zeiss (1 mentions)
Endothelial cell growth medium 2, by PromoCell (1 mentions)
Easysep human monocyte enrichment kit, by STEMCELL (1 mentions)
41 protocols using aim 5 serum free medium
1
Inflammasome Activation by Viral Infections
2
In Vitro Angiogenesis Assay
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To evaluate the angiogenic potential of the samples, a tube formation assay was performed. A matrigel (reduced in growth factors; 354230, BD Biosciences; Olen, Belgium) was thawed on ice overnight, and pipette tips and angiogenesis slides (81506, ibidi GmbH, Gräfelfing, Germany) were precooled at 4 °C. An amount of 10 µL of matrigel was pipetted in every well of angiogenesis µ-slides and incubated for 1 h at 37 °C. HUVECs were cultured and counted as already described. Cells were resuspended in basal Endothelial Cell Growth Medium 2 without growth factors (PromoCell GmbH, Heidelberg, Germany), and 10,000 cells/well were added to the matrigel. Then, 40 µL of the supernatant, obtained from cell culture experiments in 3D collagen sheets, were added to each well. Endothelial Cell Growth Medium 2 (PromoCell GmbH, Heidelberg, Germany) served as positive control, and AIM V™ serum-free medium (12055091, Thermo Fisher Scientific, Waltham, MA, USA) served as a negative control. It was mixed gently and incubated in a controlled atmosphere of 95% humidity, 5% CO2 at 37 °C for 4 h. Afterward, two light microscope images were taken per well with Zeiss Axiovert 25 (Zeiss AG, Oberkochen, Germany) and angiogenic potential was analyzed with the software Wimasis (Onimagin Technologies SCA, Cardoba, Spain).
3
Enriching Monocytes and T Cells from PBMCs
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Viably cryopreserved peripheral blood mononuclear cells (PBMCs) specimens (4–6 million cells) were thawed using AIM-V Serum Free Medium (Thermo Fisher) supplemented with 2% DNase, washed, and resuspended in buffer consisting of PBS, 3% BSA, and 1 mM EDTA. An aliquot of all PBMCs were stained and quantified using the Countess Automated Cell Counter (Life technologies). PBMCs were used to negatively select for monocytes (CD14+) by magnetic bead separation (EasySep Human Monocyte Enrichment Kit without CD16 depletion) or T cells (EasySep Human CD8+ T Cell Enrichment Kit) according to manufacturer’s instructions (StemCell Technologies). DNA and RNA were isolated from enriched monocytes or T cells using the AllPrep DNA/RNA kit (Qiagen) according to the manufacturer’s recommendations for cells. Nucleic acid concentrations were determined using the Qubit DNA Broad Range or RNA Broad Range fluorescence assays (Life Technologies) and Qubit Instrument (Life Technologies).
4
Sorafenib Dosing in Vitro
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In vitro dosing: 0.1, 1, 5, 10 µM of sorafenib (Bayer, Leverkusen, Germany) were prepared from 1 mM stock solution stored in DMSO at −80 °C. Final concentrations of drug were prepared in AIM-V serum free medium (Thermo Fisher Scientific, Grand Island, NY, USA) and repeated freeze-thaws avoided.
5
PBMC Isolation from Peripheral Blood
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Blood samples were collected in BD Vacutainer® sodium heparin tubes (BD, Le Pont de Claix, France), kept at room temperature and processed within 3–4 h. Peripheral Blood Mononuclear Cells (PBMC) were obtained from peripheral blood diluted 1:2 in AIM V® Serum Free Medium (Thermo Fisher Scientific, Waltham, USA) by using Histopaque®-1077 (Sigma Aldrich, St Louis, USA) following the provider’s recommendations. After centrifugation (400×g, 30 min, + 20 °C), plasma was collected, aliquoted and kept at − 20 °C for antibody and metabolomics analysis. PBMC were collected, washed several times with PBS-EDTA 2 mM and finally suspended in RPMI-1640 medium supplemented with 5% autologous plasma, 2 mM l -glutamine, 100 U penicillin, 100 µg/mL streptomycin (All from GIBCO®, Thermo Fisher Scientific, Waltham, USA) for cellular analysis.
6
Transmigration Assay for CAF-Mediated T Cell Recruitment
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CAFs (3.0 × 103 cells) in 50 µL of FGM2 were seeded into the upper chamber of a Transwell plate (Corning HTS Transwell 96 wells, #CLS3388, Corning Inc.), and 200 µL of FGM2 was added to the lower well. After 24 h, CAFs were transfected with siRNAs as described above and incubated for 48 h at 37 °C. Medium in the upper chamber was subsequently replaced with 50 µL of AIM V serum-free medium (Thermo Fisher Scientific) with 10% human serum (Biowest, Nuaillé, France) containing 1 × 105 CD8+ T lymphocytes. The medium in the lower well was replaced with 200 µL of culture medium with 10% human serum and recombinant human CXCL10 (1 ng/mL, Shenandoah Biotechnology, Warminster, PA, USA). After incubation for an additional 24 h, cells in the lower chamber were counted using a Countess C10281 automated cell counter (Thermo Fisher Scientific).
7
Exosome Isolation and Characterization
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The cells were grown to approximately 80% confluence, washed three times with phosphate-buffered saline (PBS), and cultured in AIM V serum-free medium (Thermo Fisher Scientific) for 2 or 3 days. Exosomes were collected from the medium by centrifugation. In brief, the medium was collected and sequentially centrifuged at 300×g for 10 min, 2000×g for 20 min, and 10,000×g for 30 min, and then filtered through a 0.22 μm Millex-GV filter (Merck Millipore, Burlington, MA, USA) to remove cells, cellular debris, and large EVs. The medium was then centrifuged at 210,000×g for 70 min using a Beckman L-70K ultracentrifuge (Beckman Instruments, Brea, CA, USA) with an SW 41 Ti swinging-bucket rotor (Beckman Instruments) [24 (link)]. The supernatant was discarded, and the pellet was washed twice with PBS. The pellet was resuspended in PBS and stored at −80 °C. Protein content was measured using a micro BCA protein assay (Thermo Fisher Scientific). The exosomes were characterized using tunable resistive pulse sensing with a qNano instrument (Meiwafosis Co., Tokyo, Japan) according to the manufacturer's instructions.
8
Chemically Defined Serum-free Media Comparison
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The following chemically defined or partially defined serum-free media were used: X-Vivo15 Hematopoietic Cell Medium (X-Vivo15) (Lonza, Bend, OR), Macrophage-SFM (Thermo Fisher Scientific, Waltham, MA), AIM V Serum-Free Medium (AIM V) (Thermo Fisher Scientific); and ImmunoCult-XF T Cell Expansion Medium (ImmunoCult-XF) (STEMCELL Technologies, Vancouver, BC, Canada). Control comparisons were performed using RPMI 1640 medium with l -glutamine and phenol red (Thermo Fisher Scientific) or 1× calcium- and magnesium-free PBS (Sigma Aldrich, St. Louis, MO).
9
Evaluating Nr4a1 and Treml4 in PECs
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PECs were harvested from untreated wild-type B6 and Nr4a1−/− mice and allowed to adhere to plastic wells (6-well plates, 2 × 106 cells/well) for 1 hr in AIM V serum free medium (Thermo Fisher Scientific, Waltham, MA). Non-adherent cells were washed off with PBS and the adherent cells were cultured for 3 hr in complete DMEM + 10% FBS medium containing LPS (1 µg/ml in PBS) or PBS alone. Nr4a1 and Treml4 expression was quantified by qPCR as above.
10
Chemically Defined Serum-free Media Comparison
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