We used buffalo Sertoli cells that were treated with 1 μg/μL Mitomycin C (HaiZheng, China) for 2 h as a feeder layer. The SSC-like cells we collected were seeded on the feeder layer and incubated in IMDM culture medium with the known component KSR (Gibco) or the unknown components (FBS), fetuin (Sigma, USA), MEM Non-Essential Amino Acids Solution (10 μL/mL, 100×; Gibco), Lipid mixture 1, Chemically defined (10 μL/mL; Sigma), glial cell-derived neurotrophic factor (GDNF, 20 ng/mL; ProSpec, Israeli), GFRα1 (100 μg/mL; R&D Systems, USA), bFGF (fibroblast growth factor-2, 10 ng/mL, ProSpec), β-Me (2-hydroxy-1-ethanethiol, 0.1 mM, Sangon Biotech, China), B27 (20 μL/mL, Gibco) and LIF2010 (10 U/mL, Millipore, Germany) [14 (link)]. The cells were cultured in a 5% CO2 atmosphere at 37°C, and the culture medium was changed every 2 days. The cells were passaged every 4 days.
Lipid mixture 1
Manufactured by Merck Group
Sourced in United States, Canada
Lipid Mixture 1 is a laboratory product offered by Merck Group. It is a mixture of various lipids, designed for use in research and experimental applications. The core function of this product is to provide a standardized and consistent source of lipids for use in scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque plus, by GE Healthcare (4 mentions)
Stemspan sfem medium, by STEMCELL (4 mentions)
Human cd34 microbead kit, by Miltenyi Biotec (3 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Il 21, by Thermo Fisher Scientific (2 mentions)
7 aad, by BD (2 mentions)
Cd19 pe lt19, by Miltenyi Biotec (2 mentions)
Cd138 allophycocyanin b b4, by Miltenyi Biotec (2 mentions)
Cd38 af700, by BioLegend (2 mentions)
Cd20 efluor v450 2h7, by Thermo Fisher Scientific (2 mentions)
Cd27 af647 lt27, by Bio-Rad (2 mentions)
Cd19 percp cy5.5 sj225c1, by BD (2 mentions)
Cd24 fitc ml5, by BD (2 mentions)
Cd27 bv605, by BioLegend (2 mentions)
Cd3viogreen bw264 56, by Miltenyi Biotec (2 mentions)
Unconjugatedgoat anti irf4 m 17, by Santa Cruz Biotechnology (2 mentions)
Donkey anti goat igg af488, by Thermo Fisher Scientific (2 mentions)
Hybridomax hybridoma growth supplement, by Gentaur (2 mentions)
Mem amino acids solution, by Merck Group (2 mentions)
Ifn α, by Merck Group (2 mentions)
18 protocols using lipid mixture 1
1
Sertoli Cell-Derived Feeder Layer Protocol
2
Multiparametric Flow Cytometry of B Cells
3
Comprehensive Multicolor Flow Cytometry for Plasma Cell Analysis
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Abs used were CD19 PE (LT19; Miltenyi Biotec), CD138 allophycocyanin (B-B4; Miltenyi Biotec), CD38 PE-Cy7 (HB7; BD Biosciences), CD38 AF700 (HIT2; BioLegend), CD20 eFluor V450 (2H7; eBioscience); CD27 AF647 (LT27; AbD Serotec), CD27 FITC (M-T271), CD19 PerCP-Cy5.5 (SJ225C1; BD Biosciences), CD19 PE-Cy7 (SJ225C1; BD Biosciences), CD24 FITC (ML5; BD Biosciences), CD84 PE (CD84.1.21; BioLegend), CD38 PerCP-Cy5.5 (HIT2; BD Biosciences), CD95 BV421 (DX2; BioLegend), CD20 allophycocyanin-H7 (L27; BD Biosciences), CD27 BV605 (O323; BioLegend), CD3 VioGreen (BW264/56; Miltenyi Biotec), Ki67 FITC (B56; BD Biosciences), unconjugated goat anti-IRF4 (M-17; Santa Cruz Biotechnology), and donkey anti-goat IgG AF488 (polyclonal; Invitrogen). Controls were isotype-matched mouse mAbs. Annexin V FITC was from eBioscience, and 7-AAD was from BD Biosciences.
Reagents included human IL-2 (Roche), IL-6 (PeproTech), IFN-α (Sigma), IL-21 (PeproTech), goat anti-human F(ab′)2 fragments (anti-IgM and anti-IgG; Jackson ImmunoResearch), HybridoMax hybridoma growth supplement (Gentaur), Lipid Mixture 1, Chemically Defined (200×), and MEM Amino Acids Solution (50×, Sigma).
Reagents included human IL-2 (Roche), IL-6 (PeproTech), IFN-α (Sigma), IL-21 (PeproTech), goat anti-human F(ab′)2 fragments (anti-IgM and anti-IgG; Jackson ImmunoResearch), HybridoMax hybridoma growth supplement (Gentaur), Lipid Mixture 1, Chemically Defined (200×), and MEM Amino Acids Solution (50×, Sigma).
4
Lipid Uptake Regulation by SLC27A2
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Cells were cultured on 15 mm glass-bottom dishes. Cells were starved for 6 h in serum-free medium to assess lipid uptake under intervention or inhibition of SLC27A2. The control group, the shSLC27A2 group, and the drug-treated group were then cultured in DMEM with 2% FBS and 1:100 Lipid Mixture 1 (L0288, Sigma-Aldrich, USA). Either DMSO or Lipofermata was added for 24 h. Prior to fixation with 4% (v/v) paraformaldehyde, the cells were incubated with 1 μM BODIPY 558/568 C-12 (D3835, Invitrogen, USA) for 2 h. The cells were permeabilized with 0.1–0.2% (v/v) TritonX-100 (ST795, Beyotime, China) in PBS for 10 min, followed by washing and blocking with 5% (w/v) bovine serum albumin (BSA) for an additional hour. The fixed cells were incubated with the primary antibody SLC27A2 (1:500, sc-393906, Santa Cruz, USA) overnight at 4 °C. The next day, we washed the cells and incubated them with FITC-labeled secondary antibodies (1:100, Boster, China) for 1 h at ambient temperature. DAPI (C1002, Beyotime, China) was used to stain the nuclei. Subsequently, the images were captured using a fluorescence confocal microscope from Olympus, Japan.
5
Lipid-Defined Serum Substitute
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Example 5
Replacement of Serum with a Lipid-Defined Substitute
The lipid components in serum are tested to determine whether they could be replaced with a defined set of lipids. Charcoal stripping removes non-polar lipophilic materials (viruses, growth factors, hormones) from serum. In comparison to control conditions where CHO cells are grown in 10% FBS, CHO cells grown in charcoal-stripped FBS do not secrete Wnt3a. These data support the conclusion that a lipophilic component of serum is required for Wnt3a secretion. Supplementation with Lipid Mixture 1 (Sigma) containing cholesterol, tocopherol, and non-animal derived fatty (inoleic, linolenic, myristic, oleic, palmitic and stearic) acids will improve CHO cell growth rate and will restore Wnt3a secretion to CHO cells.
6
Maf-DKO macrophage metabolism study
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MafB/c-Maf double deficient (Maf-DKO) macrophages were a kind gift from Dr. Michael H. Sieweke (Center d'Immunologie de Marseille-Luminy). Maf-DKO cells were grown in DMEM containing 20% L929-conditioned medium and 10% FCS. BMDM were differentiated from bone marrow cells obtained from C57BL6 mice in the presence of DMEM containing 20% L929-conditioned medium and 10% FCS. Maf-DKO cells were predominantly octaploid (8c) whereas BMDM were diploid (2c) and octaploid (8c) as determined by DNA quantification with DAPI staining (data not shown). For experiments, cells were incubated for 24 h at 37°C and 5% CO2 in low glucose, low glutamine medium (henceforth LGLG medium) which contained DMEM, 44 mM sodium bicarbonate, 10% FCS, glucose (4.8 mM), and glutamine (0.8 mM) with or without IFNγ 10 ng/mL or IL-4 10 ng/mL (both from Preprotec), and C75, etomoxir, triacsin, oligomycin, DETA/NO, or SEITU (all from Sigma) at the indicated concentrations. In a set of experiments, FCS was replaced with delipidated serum (Lipoprotein deficient serum from fetal calf, Sigma) plus a 1:100 dilution of lipid mixture (Lipid mixture 1, Sigma) containing cholesterol, and arachidonic, linoleic, linolenic, myristic, oleic, palmitic, and stearic acids.
7
Lentiviral Transduction of Human HSPCs
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Cord blood of healthy donors was obtained from Cincinnati Children’s Hospital Medical Center (CCHMC, Cincinnati, USA) and subjected to isolation of mononuclear cells (MNCs) using Ficoll-Paque PLUS (GE Healthcare Life Sciences). Human CD34+ hematopoietic stem/progenitor cells (HSPCs) were then purified from MNCs by using human CD34 MicroBead Kit (Miltenyi Biotec, Auburn, CA). The CD34+ cells were cultured in StemSpan™ SFEM medium (StemCell Technologies, Vancouver, Canada) supplemented with 1% Lipid Mixture 1 (L0288, Sigma-Aldrich, St. Louis, MO), 2 mmol/L L-glutamine, 1% penicillin-streptomycin, 100 ng/mL SCF, and 2 ng/mL IL-3. Cells were infected with concentrated lentiviral particles through two rounds of “spinoculation”.
8
Serum Lipid Replacement for Cell Culture
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Example 5
Replacement of Serum with a Lipid-Defined Substitute
The lipid components in serum are tested to determine whether they could be replaced with a defined set of lipids. Charcoal stripping removes non-polar lipophilic materials (viruses, growth factors, hormones) from serum. In comparison to control conditions where CHO cells are grown in 10% FBS, CHO cells grown in charcoal-stripped FBS do not secrete Wnt3a. These data support the conclusion that a lipophilic component of serum is required for Wnt3a secretion. Supplementation with Lipid Mixture 1 (Sigma) containing cholesterol, tocopherol, and non-animal derived fatty (inoleic, linolenic, myristic, oleic, palmitic and stearic) acids will improve CHO cell growth rate and will restore Wnt3a secretion to CHO cells.
9
Modulation of Biglycan Expression in Adipocytes
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Discarded subcutaneous tissues were obtained from the transverse rectus abdominis musculocutaneous flap of nine separately recruited women (BMI = 23.4 ± 2.3 kg/m2; age = 43.1 ± 7.0 years) undergoing breast reconstruction surgery. For isolation of human preadipocytes, the SV cell fraction was re-suspended in erythrocyte lysis buffer (154 mM NH4Cl, 10 mM KHCO3; and 0.1 mM EDTA, pH 7.4) and centrifuged at 250 g for 10 min. After washing, the cells were suspended in DMEM supplemented with 10% fetal bovine serum (FBS) and used for cell culture at passage 2 to eliminate non-preadipocyte cell contamination. Preadipocytes were differentiated into adipocytes as previously described34 (link).
To investigate the effects of proinflammatory cytokines and ER stressors on biglycan expression, human preadipocytes and fully differentiated adipocytes were incubated in serum-free medium for 24 h with TNF-α, IL-1β, tunicamycin, thapsigargin, or homocysteine. The effects of cell incubation with palmitate, oleate, or lipid mixture 1 (product number L0288; Sigma, St. Louis, MO; its lipid content consists of 2 μg/ml arachidonic acid, 10 μg/ml of each of linoleic, linolenic, myristic, oleic, palmitic, and stearic acid, and 0.22 mg/ml cholesterol) were also examined. Biglycan mRNA levels were measured in cell lysates with qPCR.
To investigate the effects of proinflammatory cytokines and ER stressors on biglycan expression, human preadipocytes and fully differentiated adipocytes were incubated in serum-free medium for 24 h with TNF-α, IL-1β, tunicamycin, thapsigargin, or homocysteine. The effects of cell incubation with palmitate, oleate, or lipid mixture 1 (product number L0288; Sigma, St. Louis, MO; its lipid content consists of 2 μg/ml arachidonic acid, 10 μg/ml of each of linoleic, linolenic, myristic, oleic, palmitic, and stearic acid, and 0.22 mg/ml cholesterol) were also examined. Biglycan mRNA levels were measured in cell lysates with qPCR.
10
Cultivation of Primary ccRCC Cells
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Fresh tumor tissue from eight patients was obtained and cultivated as reported before (16 (link)). All patients underwent partial or complete nephrectomy for ccRCC at Bonn University Hospital (Table 1
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