Recombinant mouse C3a, TNF-α and IL-6 were purchased from Novoprotein. In addition, Recombinant mouse C3a from R&D Systems was used in some of the experiments as indicated. C3a was used for primary astrocyte stimulation at the indicated concentrations; TNF-α and IL-6 were administered to MB cells at lower (200 ng/mL) and higher (500 ng/mL) concentrations, and the data shown in the results are representative of the 500 ng/mL treatment groups. C3aR antagonist (SB290157), p38 inhibitor (SB203580) and TNF-α receptor antagonist (R-7050) were all purchased from ApexBio. SB290157 and SB203580 were used at 2 μM for astrocyte administration, and R-7050 was used at 500 nM for MB cell administration in vitro.
Interleukin 6 (il 6)
Manufactured by Novoprotein
Sourced in China
IL-6 is a recombinant protein produced in E. coli. It is a cytokine that plays a critical role in immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Stem cell factor (scf), by Novoprotein (4 mentions)
Tnf α, by Novoprotein (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Stempro 34 medium, by Thermo Fisher Scientific (3 mentions)
Nutrient supplement, by Thermo Fisher Scientific (3 mentions)
Gm csf, by Novoprotein (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Il 23, by Novoprotein (2 mentions)
Sb203580, by Apexbio (1 mentions)
Recombinant mouse c3a, by R&D Systems (1 mentions)
Macs multistand, by Miltenyi Biotec (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Midimacs separator, by Miltenyi Biotec (1 mentions)
Interleukin 4 (il 4), by Novoprotein (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Defined keratinocyte serum free medium, by Thermo Fisher Scientific (1 mentions)
Mycoplasma contamination, by Qiagen (1 mentions)
Newborn cow serum, by Thermo Fisher Scientific (1 mentions)
13 protocols using interleukin 6 (il 6)
1
Astrocyte and Medulloblastoma Cell Stimulation
2
Generating Mature Dendritic Cells
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Monocytes were isolated by CD14 MicroBeads (Miltenyi) using MACS multistand and MidiMACS separator (Miltenyi). Expression of CD14 was analyzed by flow cytometry and purity of the CD14+ cells was >95%. Purified CD14+ cells were then cultured in RPMI‐1640 medium containing 10% FBS, 100 U/ml penicillin, and 100 ng/mL streptomycin at the concentration of 1%, supplemented with 40 ng/ml GM‐CSF (Novoprotein) and 20 ng/mL IL‐4 (Novoprotein). Cells were incubated at 37°C and 5% CO2 for 7 days. The fresh medium was replaced gently every 3 days. DCs were matured for another 48 h by adding 10 ng/mL TNF‐α, 1000 U/mL IL‐6, and 1 mg/mL PGE2 (both from Novoprotein).
3
Characterization of EBV-negative NPC Cell Lines
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Five EBV-negative NPC cell lines, namely CNE1, CNE2, HONE1, 5–8F, 6–10B, were generously provided by Prof. Musheng Zeng (Sun Yat-sen University Cancer Center, China). The HK1-EBV and two immortalized normal human nasopharyngeal epithelial cell lines NP460hTert-EBV and NP460hTert were provided by Prof. George S.W. Tsao, University of Hong Kong, China. The cell lines tested negative for mycoplasma contamination (Qiagen, Germany). The NPC cell lines were cultured in RPMI-1640 (Invitrogen, USA) supplemented with 10% newborn cow serum (Gibco, USA); NP460hTert-EBV and NP460hTert, defined keratinocyte serum‐free medium (Invitrogen); and 293T cells, DMEM (Invitrogen) supplemented with 10% newborn cow serum (Gibco). The cell lines were incubated in a humidified chamber with 5% CO2 at 37 °C. All gifted cell lines were authenticated by STR profile. Barflomycin A1 (Baf-A1; Selleckchem, USA), Stattic (Selleckchem), and IL-6 (Novoprotein, USA) were dissolved according to the manufacturers’ instructions. The antibodies used for subsequent experiments are listed in Table S4 .
4
In Vivo HPSC Transduction and Transfer
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For in vivo validation, plasmids encoding positive shRNA hits were modified to delete the puromycin resistance gene, amplified using an endotoxin-free DNA purification kit (12362, Qiagen), and packaged to generate retroviral supernatants as described above. For HPSC enrichment, wild type C57BL/6J mice were treated with 4 mg/mouse 5-fluorouracil (5-FU) (Sigma) by intravenous (iv) injection, followed by bone marrow cell collection 5 days later. HPSCs were stimulated with SCF (100ng/ml, Novoprotein), IL-3 (20ng/ml, Novoprotein) and IL-6 (25ng/ml, Novoprotein) for 36 hours before transduction. Retrovirus transduction of HPSCs was performed by spinoculation (2000rpm, 2 hours without brake) and media was replaced 4 hours later. Transduced cells were harvested after 20 hours for intravenous transfer into IgMb-macroself mice. Alternatively, freshly isolated BM cells from mutant mice were depleted of red blood cells using ACK lysis buffer, counted, and resuspended in PBS. 1-10x106 retrovirally transduced HPSCs or freshly isolated BM cells were iv injected into recipient mice. IgMb-macroself mice were irradiated with 6 Gy by X-Ray before reconstitution. Recipient mice were euthanized and their splenocytes analyzed by flow cytometry 8 weeks after reconstitution.
5
STAT3-Dependent Luciferase Assay
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After transfected with STAT3-Luc or empty vector along with the internal control renilla luciferase by Lipofectamine® 2000 (Invitrogen) for 24 h, TE13 cells were incubated with indicated agents for 12 h, and then stimulated with 50 ng/ml IL-6 (Novoprotein, Shanghai, China) or vehicle control for 20 min. Cells were then prepared for luciferase assay by using Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA) as described previously [21 (link), 22 (link)].
6
LAD2 Cell Degranulation Assay
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LAD2 cells were grown in StemPro-34 medium (Gibco) supplemented with 100 μg/mL SCF (Novoprotein), 100 μg/mL IL-6 (Novoprotein), nutrient supplement (NS) (Gibco), 100 U/mL penicillin (Invitrogen), 100 μg/mL of streptomycin (Invitrogen) and 2 mM L-Glutamine (Gibco) at 37°C under 5% CO2. LAD2 cell degranulation was evaluated by measuring the release of β-hexosaminidase (Hsieh et al., 2019 (link)). Briefly, LAD2 cells (3.5 × 105) were exposed to HIV-JRFL/VLP (10 or 100 ng p24Gag) or HIV-HXB2/VLP (10 or 100 ng p24Gag) for the indicated times. C48/80 (4 μg/mL) was used to induce MC degranulation as the positive control. For measuring β-hexosaminidase activity, the substrate of p-nitrophenyl-N-acetyl-β-D-glucosaminide was dissolved in 0.1 M sodium citrate (pH 4.5) for reaction for 1 h at 37°C, then 0.1 M carbonate buffer (pH 10) was added to stop the reaction. The product of 4-p-nitrophenyl was detected at absorbance of 405 nm. β-hexosaminidase was measured in the supernatant as well as the cell lysate solubilized in 0.1% Triton-X100. Percentage of degranulation was calculated by dividing the absorbance in supernatant by the sum of absorbance in both supernatant and cell lysate.
7
Meningitic E. coli Infection of hBMECs
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Meningitic E. coli strain infection of primary hBMECs, as well as U251 cells and HUVECs, was performed in accordance with previously described methods61 62 (link). Briefly, E. coli overnight culture was resuspended and diluted in serum-free medium and added to the starved confluent primary hBMEC monolayer grown in 10-cm dishes at a multiplicity of infection of 10 (approximately 108 colony-forming units per dish) to allow invasion at 37 °C for 3 h. For cytokine stimulation, recombinant human IL-8, MIP-2, GRO-α, IL-1β, IL-6 and TNF-α were purchased from Novoprotein Scientific (Shanghai, China) and used at a final concentration of 10 ng/mL to stimulate the primary hBMECs for 24 h. Finally, cells were washed three times with chilled PBS and subjected to RNA extraction by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions.
8
Immunosuppressive Ability Assessment of BM-derived CD11b+Ly6G+Ly6C low Cells
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(1) Cell lines. ID8 mouse ovarian cancer cells were donated by Professor Dong Chen (Institute of Immunology, Tsinghua University), and B16F10 mouse melanoma cells were purchased from the Cell Bank of the Shanghai Branch of the Chinese Academy of Sciences. Cells were cultured in high-glucose DMEM (Corning) supplemented with 10% FBS (Corning) and 1% penicillin-streptomycin (Haoyang Biological Manufacture). All cells were cultured in a 37°C, 5% CO2 cell incubator. (2) Activated T cells. We collected WT mouse spleen-derived T cells and labeled with CFSE. Then, these T cells were activated by anti-CD3 and anti-CD28 antibodies (BioLegend). (3) Assessment of the immunosuppressive ability of BM-derived CD11b+Ly6G+Ly6 Clow cells. WT mice BM-derived CD11b+Ly6G+Ly6Clow cells with or without 100 ng/mL mouse GM-CSF (Novoprotein, Shanghai, China) and 100 ng/mL mouse IL-6 (Novoprotein) stimulation cocultured with activated T cells for 96 hours. The FITC signal intensity of T cells was detected by FCM and the concentration of IL-2 in the supernatants was determined by a commercial ELISA kit (Proteintech, Wuhan, China). (4) Establishment of the primary PMN-MDSCs culture system in vitro. Primary mouse BM-derived CD11b+Ly6G+Ly6Clow cells obtained by the method described above were cultured with 100 ng/mL GM-CSF (Novoprotein) and 100 ng/mL IL-6 (Novoprotein) and used in subsequent experiments.
9
Pseudotyped HIV Infection of Brain Cells
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Human brain microvascular endothelial cells hCMEC/D3 (purchased from Meisen CTCC, Zhejiang, China) and microglial cells HCM3 (purchased from Meisen CTCC, Zhejiang, China) were cultured in DMEM medium (Gibco, Invitrogen, California, USA) containing 10% fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin. Human mast cells LAD2 were cultured in RPMI 1640 medium (Gibco), containing 10% fatal FBS with 100 U/mL penicillin and 100 μg/mL streptomycin. For degranulation, LAD2 cells (purchased from Huzhen Company, Shanghai, China) were grown in StemPro-34 medium (Gibco) supplemented with 100 μg/ml stem cell factor (Novoprotein, Suzhou, China), 100 μg/ml IL-6 (Novoprotein), nutrient supplement (Gibco), 100 U/ml penicillin (Invitrogen, California, USA), 100 µg/ml of streptomycin (Invitrogen), and 2 mM L-Glutamine (Gibco).
HIV-NL4-3/spike pseudotyped virus was generated by Lipo 2000 transfection reagent (Invitrogen)-mediated co-transfection of HEK293T cells, with the spike-expressing plasmid pcDNA3.1-2019-nCoV-S-IRES (strain 2019-nCoV WIV04) and pNL4-3. Luc. ΔR ΔE. These two plasmids and HEK293T cells are provided by Dr. Lu Lu (Fudan University, Shanghai, China). Harvested HIV-NL4-3/spike pseudovirus were used to infect hCMEC/D3 and HMC3 cells for 48h, viral infection was measured by detecting luciferase activity (Promega, Madison, WI, USA).
HIV-NL4-3/spike pseudotyped virus was generated by Lipo 2000 transfection reagent (Invitrogen)-mediated co-transfection of HEK293T cells, with the spike-expressing plasmid pcDNA3.1-2019-nCoV-S-IRES (strain 2019-nCoV WIV04) and pNL4-3. Luc. ΔR ΔE. These two plasmids and HEK293T cells are provided by Dr. Lu Lu (Fudan University, Shanghai, China). Harvested HIV-NL4-3/spike pseudovirus were used to infect hCMEC/D3 and HMC3 cells for 48h, viral infection was measured by detecting luciferase activity (Promega, Madison, WI, USA).
10
Modulation of IGF-1 and IL-6 Signaling
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Multiple myeloma cell lines were maintained overnight in Iscove’s modified Dulbecco’s medium containing 0.5% fetal bovine serum, and treated with 100 μM of S14161 or BENC-511 for 0.5 to 2 hours before stimulation with 100 ng/mL human recombinant insulin-like growth factor-1(IGF-1, PeproTech, Rocky Hill, NJ) or 50 ng/mL of interleukin-6 (IL-6, Novoprotein, Summit, NJ) for 15 minutes before being lysed in a RIPA buffer containing 1 mM orthovanadate [11 (link)]. After clarification, cell lysates were subjected to Western blotting analysis with anti-phospho-AKT (S473) or anti-AKT.
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