Human glioma tissues and NBTs were collected from patients at the Department of Neurosurgery of Shengjing Hospital of China Medical University (n = 5). All of the tissue samples were immediately frozen in liquid nitrogen after surgical resection and stored at −80°C until use. Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of Shengjing Hospital of China Medical University. Glioma tissue samples were divided into two groups, low-grade glioma tissues (LGGTs; n = 16) and HGGTs (n = 10), by experienced neuropathologists according to the 2007 World Health Organization (WHO) classification of tumors in the CNS. HA cells were obtained from Scien-Cell Research Laboratories (Carlsbad, CA, USA) and grown in RPMI-1640 culture medium (GIBCO, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; GIBCO, Carlsbad, CA, USA). Human glioma cell lines (U87 and U251) and HEK293T cells were purchased from Shanghai Genechem and grown in DMEM/high glucose with 10% FBS. All cells were maintained in a humidified incubator at 37°C with 5% CO2.
Hek293t cells
Manufactured by Genechem
Sourced in China
HEK293T cells are a widely used human embryonic kidney cell line. They are a popular model system for various cell biology, molecular biology, and biotechnology applications due to their high transfection efficiency and ability to express recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Ha cells, by ScienCell (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hek293t, by Genechem (1 mentions)
Lipofectamine 2000, by Genechem (1 mentions)
Murine raw264.7 macrophages, by American Type Culture Collection (1 mentions)
Lentivirus package plasmids, by Genechem (1 mentions)
7 protocols using hek293t cells
1
Collection and Cultivation of Glioma Samples
2
Lentiviral Knockdown of ccny
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Recombinant lentiviral vectors were designed and packaged in HEK293T cells by GeneChem (Shanghai, China). The sgRNA sequence designed based on the human ccny mRNA (NM_181698) sequence was 5ʹ-TGAATGTGCCATCGTCACCC-3ʹ, 5ʹ-TGAACAGTGTCCGAACGAAC-3ʹ, 5ʹ-TAGATATCTGTCCGGCCAAC-3ʹ.
For cellular infection, cells were cultured at 6000 cells/well in 96-well culture plate and infected with recombinant lentivirus 24 hrs later. Stable clones were selected and identified by Western blot.
For cellular infection, cells were cultured at 6000 cells/well in 96-well culture plate and infected with recombinant lentivirus 24 hrs later. Stable clones were selected and identified by Western blot.
3
Generating Stable SphK1/2 Knockdown Cells
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The GV369 lentiviral vectors encoding SphK1 shRNA and SphK2 shRNA were synthesized by Shanghai Genechem Co (Shanghai, China). The construct, together with the lentivirus Helper plasmids, was co-transfected to HEK-293T cells (Genechem). The generated lentivirus were added to prostate cancer cells (cultured in polybrene medium) for 24 h. Cells were then cultured in puromycin (5 μg/mL)-containing complete medium for another 48 h, and stable cells established. SphK1/2 expression in stable cells was tested by Western blotting and qRT-PCR assays. The scramble control shRNA lentivirus (Genechem, Shanghai, China) was added to the control prostate cancer cells.
4
Lentiviral Transduction of HEK293T Cells
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Human embryonic kidney 293T cells (HEK293T) were used for lentiviral packing. HEK293T cells was provided by Genechem Co., Ltd. (Shanghai, China) and originally purchased from American Type Culture Collection (ATCC, Manassas, VA, CRL-3216). The cells were cultured in 10 cm dishes for 2–3 days until they reached 90–95% confluency. The recombinant virus plasmid pLV-nol3-EGFP, which encodes the full-length nol3, and the control vector pLV-EGFP, together with packaging plasmids (pLP1, pLP2, and pLP/VSVG), were cotransfected into HEK293T cells using Lipofectamine™ 2000 (all from GeneChem Co., Ltd., Shanghai, China). After 48 h of transduction, the lentiviral particles contained in the supernatant of 293T cells were harvested and then concentrated by passing through a 0.45-μm filter. The concentrated solutions were then added to cultured BMSCs at a variety of multiplicities of infections ranging from 0 to 100. After 72 h, the transduction efficiency was assessed via fluorescence microscopy and qRT-PCR.
5
Macrophage Polarization Protocol
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Murine Raw264.7 macrophages were obtained from the American Type Culture Collection (ATCC), and HEK-293T cells were purchased from Genechem (Shanghai, China). Murine alveolar epithelial cell line MLE-12 and immortalized murine embryo fibroblasts (MEF) were kindly provided by Prof. Guanghui Jin. All cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and cultured at 37 °C and 5% CO2.
Raw264.7 cells were exposed to 20 μg/mL BLM for 48 h, and then the culture medium (CM) was subjected to centrifugation to remove the cell debris. The collected supernatant was treated with MEF. The M1 and M2 macrophages were induced as described previously [56 (link)].
Raw264.7 cells were exposed to 20 μg/mL BLM for 48 h, and then the culture medium (CM) was subjected to centrifugation to remove the cell debris. The collected supernatant was treated with MEF. The M1 and M2 macrophages were induced as described previously [56 (link)].
6
Lentiviral Transduction of miR-6862
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GV369 lentiviral constructs encoding the miR-6862 precursor sequence (premiR-6862, sequence listed in Table 2 ) or the anti-sense sequence (anti-premiR-6862) were synthesized by Genechem (Shanghai, China). Each was transfected in HEK-293T cells along with the lentivirus package plasmids (Genechem) to generate the premiR-6862 expression lentivirus (“lv-premiR-6862”) and the premiR-6862 anti-sense lentivirus (“lv-antagomiR-6862”). Neuronal cells were cultured into six well-tissue plates (at 2 × 105 cells per well)under the polybrene-containing medium, and lentivirus was then added. Puromycin (5.0 μg/mL) was further added to select stable cells, and mature miR-6862 expression (sequence listed in Table 2 ) was examined by qPCR.
7
Modulating miR-330-5p and SNHG3 in Cancer
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shRNAs binding the sequence of SNHG3 (sh-SNHG3), scrambled control (sh-control), anti-miR-330-5p (anti-miR-330), and anti-miR-control (anti-miR-con) were cloned into pCDH vectors containing red fluorescent protein (mcherry) reporter. Briefly, these plasmids were transfected into HEK293T cells, along with helper plasmids as packaging vectors (GeneChem, Shanghai, China). MD-MBA-453 cells were transduced with a lentivirus containing anti-miR-330-5p using the lentivirus and 5 mg/ml polybrene Genechem (Shanghai, China). FACS analysis was performed 2 days after the transfection with the lentivirus for measuring mCherry signal to obtain cells with stably miR-330-5p knockdown. Similarly, breast cancer patient–derived CAFs were transduced with sh-SNHG3 expressing lentivirus to obtain stably SNHG3 knockdown cells.
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