RAW264.7 cells were obtained from China Center for Type Culture Collection (Wuhan, China). All cell lines have been authenticated using STR profiling and tested for mycoplasma contamination. RAW264.7 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. BMDCs were harvested after 7 days of culture with GM-CSF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA) and IL-4 (10 ng/mL; PeproTech), and BMDMs with M-CSF (50 ng/mL, PeproTech). The purity of BMDCs and BMDMs was more than 90% assessed by flow cytometry. Atg7-knockdown BMDCs (Atg7 KD) were established by siRNA (Ribobio) transfection using riboFECT reagent (Ribobio). LPS (0111:B4, L3024, Sigma-Aldrich), FITC-LPS (0111:B4, F3665, Sigma-Aldrich), z-VAD-fmk (HY-16658B, MedChemExpress), Erastin (E7781, Sigma-Aldrich), Ferrostatin-1 (SML0583, Sigma-Aldrich), Rapamycin (V900930, Sigma-Aldrich), Chloroquine (C6628, Sigma-Aldrich) were supplemented into culture medium respectively in the following experiments.
Ribofect reagent
Manufactured by RiboBio
Sourced in China
RiboFECT is a transfection reagent designed to facilitate the delivery of RNA molecules, such as small interfering RNA (siRNA) or microRNA (miRNA), into eukaryotic cells. It is a cationic lipid-based formulation that forms complexes with the target RNA, which can then be efficiently taken up by cells.
Automatically generated - may contain errors
Lab products found in correlation
Fitc lps, by Merck Group (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Raw264.7 cells, by National Collection of Authenticated Cell Cultures (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Ferrostatin 1, by Merck Group (1 mentions)
Erastin, by Merck Group (1 mentions)
Z vad fmk, by MedChemExpress (1 mentions)
Control sirna, by RiboBio (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
B 27 serum free supplement, by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Ribofect buffer, by RiboBio (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
4 protocols using ribofect reagent
1
Establishment and Characterization of Macrophage Cell Models
2
Isolation and siRNA Knockdown of Mouse Splenic B Cells
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B16 cells (American Type Culture Collection) were cultured in RPMI1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco) with penicillin/streptomycin (Gibco) at 37°C with 5% CO2. Splenic B cells were isolated with Magnetic Cell Sorting (MACS) beads by negative selection according to the manufacturer’s protocol (Miltenyi Biotec). The purity was routine >96% as assessed by staining with anti-CD19. For siRNA transfection, mouse Adam28 siRNA (Ribobio) or control siRNA (Ribobio) was used in the presence of riboFECT reagent (Ribobio). B cells were harvested 48 h later for qRT-PCR analysis. The following siRNAs were used: siAdam28-1: GCATGATTCATGACTACTT; siAdam28-2: GCAGTCGTGTCAATTACAA; and siAdam28-3: CACCAAGGATGCCAAGCTA.
3
In vitro Validation of circAbca1 Inhibitor
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The targeted binding relationship was preliminarily verified in vitro. Primary C57BL/6 mouse dorsal root ganglion cells (PriCells, Wuhan, China) were cultured at 37°C and 5% CO2 in Neurobasal medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% B-27™ serum-free supplement (50X, Thermo Fisher Scientific) and 100 kU/L of penicillin and streptomycin (Thermo Fisher Scientific). Cultured cells were transfected with circAbca1 inhibitor (5′-CCA ACT CAA TGT CAG CTG T-3′) or the corresponding controls (Ribobio, Guangzhou, China). Next, 120 μL of 1× riboFECT™ Buffer was diluted with 5 μL and 20 μM siRNA stock solution, and then 12 μL riboFECT™ Reagent (Ribobio, Guangzhou, China) was added and mixed at room temperature for 10 minutes. The mixture was added to medium without penicillin and streptomycin, and then added to cells for an additional 48 hours of culture.
4
Optimized siRNA Transfection and Sorafenib Treatment in Thyroid Cancer Cell Lines
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The PTC-derived cell lines TPC-1, IHH-4, K1 (Fenghui Biotechnology, China), and B-CPAP (Shanghai Cell Bank of Chinese Academy of Science, China) were used in this study. B-CPAP and IHH-4 cells were cultured in RPMI 1640, and TPC-1 and K1 cells were cultured in DMEM. The media contained 10% fetal bovine serum (Biological Industries, USA) and 100 U/mL penicillin-streptomycin (Yeasen Biotech, China) in a 5% CO2 incubator at 37°C. Three small interfering (siRNA) combinations were used for transfection when cells were at 50% con uence. 120 μL of 1×riboFECT CP Buffer was added to dilute 15μL siRNA labeled with uorescent Cy5, the negative control (NC) or siRNA-PVT1 2+3+4 per 5 μL in the TPC-1 cells and siRNA-PVT1 1+3+4 per 5 μL in the B-CPAP cells, cause through preliminary experiments we found that this can achieve the maximum knocking down e ciency. Then, 12 μL of riboFECT Reagent (RiboBio, China) was added and incubated for 15 min, and the samples were incubated in penicillin-streptomycin-free culture medium for 48 h. After transfection, sorafenib was applied at 9 μL in B-CPAP and 13 μL in TPC-1 cells according to their half maximal inhibitory concentration (IC50) values at 48 h.
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