RAW264.7 cells were cultured in DMEM medium supplemented with 10% FBS (Gibco), penicillin (50 U/mL, Gibco), and streptomycin (50 mg/mL, Gibco) at 37°C and 5% CO2. 2×105 cells were inoculated in 48 well plates on the day before stimulation, and cells were stimulated with PBS, R848 (2 µg/ml, Sigma), mouse recombinant IFN-γ (200 ng/ml, Novoprotein), iRBCs (iRBCs: cells = 3: 1), R848 plus iRBCs, IFN-γ plus iRBCs respectively for 20 h.
Ifn γ
Manufactured by Novoprotein
Sourced in China
IFN-γ is a recombinant human interferon-gamma, a cytokine that plays a crucial role in the activation of the immune system. It is commonly used as a research tool in immunology and cell biology studies.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Tnf α, by Novoprotein (3 mentions)
Il 13, by Novoprotein (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by Novoprotein (2 mentions)
Lipopolysaccharides (lps), by Novoprotein (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Abnova (1 mentions)
Hy d1056, by MedChemExpress (1 mentions)
Hy 18739, by MedChemExpress (1 mentions)
Raw264, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Basic fgf, by Thermo Fisher Scientific (1 mentions)
L glu, by Thermo Fisher Scientific (1 mentions)
Chang b, by Irvine Scientific (1 mentions)
Chang c, by Irvine Scientific (1 mentions)
19 protocols using ifn γ
1
RAW264.7 cell stimulation assay
2
Tumor Growth Modulation in Ccng2-/- Mice
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Ccng2−/− C57BL/6 mice were described previously. The TALEN-targeted Ccng2 knockout mice (Ccng2−/−) of the C57BL/6 N genetic background were generated by Cyagen (Cyagen Biosciences, Guangzhou, China) [16 (link), 19 (link)]. LLC or MC38 cells (1.5 × 106) were mixed with 3 × 105 BMDMs from wild-type (WT) or Ccng2−/− C57BL/6 mice and subcutaneously inoculated into the right flank of eight-week-old C57BL/6 mice. Each mouse was intraperitoneally injected with 2.5 μg IFN-γ (C746, Novoprotein) on Days 6, 9, and 12 after tumor cell inoculation. Tumors were periodically measured with calipers. The mice were euthanized on Day 15, and tumor weights and volumes were measured. All animal experiments were performed in accordance with relevant regulatory standards and approved by the Animal Ethics Committee of China Medical University.
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Ccng2−/− C57BL/6 mice were described previously. The TALEN-targeted Ccng2 knockout mice (Ccng2−/−) of the C57BL/6 N genetic background were generated by Cyagen (Cyagen Biosciences, Guangzhou, China) [16 (link), 19 (link)]. LLC or MC38 cells (1.5 × 106) were mixed with 3 × 105 BMDMs from wild-type (WT) or Ccng2−/− C57BL/6 mice and subcutaneously inoculated into the right flank of eight-week-old C57BL/6 mice. Each mouse was intraperitoneally injected with 2.5 μg IFN-γ (C746, Novoprotein) on Days 6, 9, and 12 after tumor cell inoculation. Tumors were periodically measured with calipers. The mice were euthanized on Day 15, and tumor weights and volumes were measured. All animal experiments were performed in accordance with relevant regulatory standards and approved by the Animal Ethics Committee of China Medical University.
3
Macrophage Polarization Modulation by HA
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RAW264.7 (provided by doc. Huang Xin) was cultured in Dulbecco's Modified Eagle Medium (DMEM, SH30022.01, Hyclone) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a 37°C incubator. Subsequently, the cells were induced with 100 ng/mL lipopolysaccharide (LPS, Sigma, L2654, USA) and 20 ng/mL interferon‐gamma (IFN‐γ, Novoprotein, CM41, China) for 24 h in a fresh medium to stimulate M1 polarization. In the presence of LPS and IFN‐γ, 20 ng/mL rhALPK1 (H00080216‐Q01; Abnova) was added to examine the effect of rhALPK1 on macrophage polarization. 2 mg/mL LMW‐HA (MACKLIN, H874944‐1g, China, molecular weight 10–100 KDa) or 2 mg/mL HMW‐HA (MACKLIN, H909939‐5g, China, molecular weight 1500–2500 KDa) was added to examine the effect of HA on macrophage polarization.
4
Macrophage Polarization Protocol
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M0 macrophages were obtained by treating THP-1 cells with 100 ng/ml phorbol 12-myristate 13-acetate (PMA, HY-18739, MedChemExpress, USA) for 48 h. Then, M0 macrophages were induced with 100 ng/ml bacterial lipopolysaccharide (LPS, HY-D1056, MedChemExpress, USA) + 2.5 ng/ml IFN-γ (C014, novoprotein, China) for 48 h to acquire the M1 phenotype. Meanwhile, M0 macrophages were induced with 10 ng/mL IL-4 (CX03, novoprotein, China) + 10 ng/mL IL-13 (CC89, novoprotein, China) for 48 h to acquire the M2 phenotype. Polarized status was confirmed utilizing flow cytometry.
5
Culture and Maintenance of Adipocyte and Macrophage Cell Lines
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Pre-adipocytes cell line 3T3-L1 and RAW264.7 cells were purchased from Stem Cell Bank, Chinese Academy of Sciences. 3T3-L1 cells were cultured with DMEM (Gibco, C11995500BT) containing 10% new bovine serum. RAW264.7 cells were cultured with RPMI-1640 (Gibco, C1187500BT) containing 10% fetal bovine serum (BI, 04-004-1A). hAMSCs were cultured in MEM-α (Gibco, A10490-01) containing 10% fetal bovine serum (Gibco, 10099-141), 2% Chang C (IrvineScientific, C108), 18% Chang B (IrvineScientific, C100), 0.1%FGF-basic (20 ng/mL, Peprotech, 100-1B), 1% L-GLU (Gibco, 35050061), and 0.1% β-Mercaptoethanol (Solarbio, Beijing, 21985023). All cells were cultured with 100 U/mL penicillin and 100 mg/mL streptomycin and maintained at 37 °C in a humidified incubator under 5% CO2. One hundred ng/mL LPS (Sigma-Aldrich, L4130), 2.5 ng/mL IFN-γ (Novoprotein, C746), and 10 μg/mL IL-4 (Novoprotein, CK74) were used in the experiments of RAW264.7 cells.
6
Isolation and Polarization of Murine Macrophages
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Euthanize C57BL/6 wild-type or Jmjd6+/− mice, and retract their abdominal skin to expose the intact peritoneal wall. For the isolation of PM, 10 mL normal saline was injected intraperitoneally, and then fluid from peritoneum was aspirated with the same syringe. After centrifuging, the peritoneal cells were collected and cultured in complete RPMI-1640 medium for 2 h to allow the adhesion of macrophages. For the isolation of BMDM, the bone marrow was harvested from their femurs and tibia, and was filtered through a 70 μm cell strainer. Cells were cultured in complete RPMI-1640 medium with 20 ng/mL M-CSF for 5 days. For tumor-supernatant treatment, PMs and BMDMs were incubated in complete RPMI-1640 medium containing 20 ng/mL M-CSF and 10% B16 conditioned media for 2 days. For classical and alternative activation of macrophages, 100 ng/mL LPS (Sigma) + 20 ng/mL IFN-γ (Novoprotein) or 20 ng/mL IL-4 (Novoprotein) were added to the culture medium for 2 days, respectively.
7
Silencing GBP5 in Jurkat Cells
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Human cell lines Jurkat, THP-1, and 293T cells were purchased from American Type Culture Collection (ATCC, United States). Cells were cultured in RPMI 1640 medium (Gibco, United States) supplemented with 10% fetal bovine serum (Gibco, United States) maintained at 37°C and 5% CO2. To knockdown GBP5 in Jurkat cells, a pool of two different siRNAs (1, 5′-GCCATAATCTCTTCATTCA-3′; and 2, 5′-GCTCGGCTTTACTTAAGGA-3′) were used. Scrambled sequence of the siRNA was used as control. RNA was synthesized at Ribo-Bio (China) and provided lyophilized. RNA was reconstituted with nuclease-free water to reach a final concentration of 20μM, before transfection using Lipofectamine RNAiMAX (Life technologies, United States) following the manufacturer’s instructions. At 48 h post-transfection, cells were stimulated with human interferon γ (IFNγ, 25μg/ml, Novoprotein, China) and lipopolysaccharides from Escherichia coli O55:B5 (LPS, 500 ng/ml, Sigma-Aldrich, United States) for 16 h before harvest.
8
Epacadostat Modulates M2 Macrophages
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THP-1 human monocytes were purchased from Wuhan Procell Life Science & Technology Co., Ltd. (Hubei, China). NSCLC cell lines (A549 and H1299) were purchased from the Institute of Life Sciences, Chinese Academy of Sciences Cell Bank (Shanghai, China). The THP-1, H1299 and A549 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin 20 μg/mL and streptomycin 20 μg/mL (Sigma, USA). All the cells were cultured in a humid environment at 37 °C and 5% CO2. In this experiment, THP-1 cells were seeded in 6-well plates, and 100 ng/mL phorbol 12-myristate 13-acetate (PMA, Abcam) was added and incubated for 48 h to differentiate the cells into M0 macrophages. LPS (100 pg/mL) and IFN-γ (20 ng/mL) or IL-4 and IL-13 (20 ng/mL) (Novoprotein, Shanghai, China) were added and incubated to differentiate the cells into M1 or M2 macrophages, respectively, for subsequent experiments. Then, Epacadostat (INCB024360, Selleck, USA) was used to treat the M2 macrophages.
9
Colon Cancer Cell Line Cultivation
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All human colon cancer cell lines, including DLD1, HCT8, WiDr, Lovo, SW48, HCT116, RKO, HCT15, and HEK293T, were all obtained from the ATCC. The mouse colon cancer cell line CT26 was obtained from the ATCC and the murine colon cancer cell line MC38 was obtained from the National Infrastructure of Cell Line Resource (Beijing, China). DMEM (Gibco, Thermo Fisher Scientific) mixed with 10% FBS (Gibco, Thermo Fisher Scientific) was used to culture all cells in a 5% CO2 atmosphere. All cell lines were validated by STR DNA finger-printing and the reauthentication took place in 2021. All cell lines were routinely tested for Mycoplasma. Cells were generally passaged 1–2 times before being used for subsequent experiments. All cell lines were passaged within 15 times in our study.
Protocols for transfection and lentivirus infection are described previously in the indicated section. After cells were transfected 48 hours with the indicated plasmid, 25-μm MG132 (Sigma-Aldrich) or DMSO (Sigma-Aldrich) was added into medium and cultured 12 hours. For IFNγ stimulation, MC38 and CT26 cells were transfected with vector and Krt17 plasmids with 50 ng/mL IFNγ (NovoProtein) or BSA (Sigma-Aldrich) for 48 hours.
Protocols for transfection and lentivirus infection are described previously in the indicated section. After cells were transfected 48 hours with the indicated plasmid, 25-μm MG132 (Sigma-Aldrich) or DMSO (Sigma-Aldrich) was added into medium and cultured 12 hours. For IFNγ stimulation, MC38 and CT26 cells were transfected with vector and Krt17 plasmids with 50 ng/mL IFNγ (NovoProtein) or BSA (Sigma-Aldrich) for 48 hours.
10
Modulating Macrophage Cytokine Release
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Primary mMφ were treated with LPS (40 ng/mL, Novoprotein) and IFN-γ (20 ng/mL, Novoprotein) for 4 h at 37 °C with 5% CO2 to induce cytokine release. For EV functional evaluation, M2-EVs or MSC-EVs (10, 20 μg/mL) were added at the same time as LPS/γ-IFN treatment. After incubation, the treated cells were collected for qPCR assay. To determine whether the results were reproducible, LPS/γ-IFN-primed macrophages were treated with different EV preparations of M2-PMφ or MSCs, and the expression of cytokines was analyzed.
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