We obtained bone marrow specimens from iron-deficiency anemia (taken as normal control), SMM, and MM patients at the Third Xiangya Hospital. All patients provided their written informed consent. Research on clinical samples was performed in accordance with the Declaration of Helsinki. All MM and SMM patients met the diagnostic criteria defined by National Comprehensive Cancer Network (NCCN). Human MM cell lines (ARP1 and RPMI-8226) were purchased from the National Collection of Authenticated Cell Cultures (China). Cells were cultured in 5% CO2 at 37 °C with RPMI-1640 medium (HyClone, USA) plus 10% fetal bovine serum (ExCell Biology, Shanghai, China) and 1% penicillin–streptomycin (HyClone, USA). The shRNA-IL5RA and shRNA-NC were synthesized by GenePharma23 (link). The cells were transfected using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. For the cell proliferation assay, myeloma cells were incubated in 96-well plates for the required time. Then, cell count kit-8 (CCK-8) reagent (Sigma-Aldrich, St Louis, Missouri; 10 µL) was added followed by incubation for 4 h. The absorbance of the solution was measured at 450 nm with a microplate imaging system.
Rpmi 8226
Manufactured by National Collection of Authenticated Cell Cultures
Sourced in China
The RPMI-8226 is a type of cell culture media designed for the growth and maintenance of human myeloma cells. It provides the necessary nutrients and growth factors to support the proliferation and survival of these specialized cell lines. The RPMI-8226 media is a widely used standard in the field of cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Mm 1s, by National Collection of Authenticated Cell Cultures (3 mentions)
Ncih929, by National Collection of Authenticated Cell Cultures (2 mentions)
Si snhg16, by Genechem (2 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Fetal bovine serum (fbs), by STEMCELL (1 mentions)
Cd138 microbeads, by STEMCELL (1 mentions)
Venetoclax, by MedChemExpress (1 mentions)
Chidamide, by MedChemExpress (1 mentions)
Oe nc, by Genechem (1 mentions)
Si nc, by Genechem (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Trypsin digestion, by Thermo Fisher Scientific (1 mentions)
Bortezomib, by Merck Group (1 mentions)
Hydroxychloroquine sulfate, by Selleck Chemicals (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Lenalidomide, by Selleck Chemicals (1 mentions)
18 protocols using rpmi 8226
1
Investigating Myeloma Cell Proliferation
2
Investigating Chidamide and Venetoclax in Multiple Myeloma
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The HMCLs ARP-1 and U266 were generously provided by Dr. Qing Yi (Center for Hematologic Malignancy, Research Institute, Houston Methodist, Houston, TX, USA), and RPMI-8226 and MM1.S were purchased from the National Collection of Authenticated Cell Cultures (Beijing, China). All cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Biological Industries, Israel) at 37 °C under 5% CO2 atmosphere.
With the approval of the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine and informed consents obtained from MM patients, we fetched bone marrow samples for the experimental study only. Primary MM cells were sorted by CD138 microbeads(STEM CELL Technologies, Canada), and then cultured in RPMI-1640 medium containing 20% FBS at 37 °C under 5% CO2 atmosphere.
Chidamide and venetoclax were purchased from Med Chem Express (New Jersey, USA).
With the approval of the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine and informed consents obtained from MM patients, we fetched bone marrow samples for the experimental study only. Primary MM cells were sorted by CD138 microbeads(STEM CELL Technologies, Canada), and then cultured in RPMI-1640 medium containing 20% FBS at 37 °C under 5% CO2 atmosphere.
Chidamide and venetoclax were purchased from Med Chem Express (New Jersey, USA).
3
Modulating miR-451 and CRNDE in Multiple Myeloma
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Human MM cell lines (MM1.S, NCIH929, U266, and RPMI-8226) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, P.R. China). MM cell lines and normal plasma cells (nPCs) were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C with a 5% CO2 atmosphere. miR-451 inhibitor and siRNAs to CRNDE were purchased from Genechem (Shanghai, P.R. China). The sequences were listed as follows: miR-451 inhibitor, 5′-AGUGACGGACGUGUUGGGCCAU-3′; si-CRNDE-1, 5′-CCATTCCATTCATTTCTCTTTCCTA-3′; si-CRNDE-2, 5′-CCTCTCATTATTCATTCCTTTCCTA-3′. Cells were transfected with the indicated nucleotides or plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions.
4
Modulating SNHG16 in MM cells
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Human MM cells RPMI-8226 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2. Overexpression of SNHG16 (OE-SNHG16) and negative control (OE-NC) vectors, siRNAs to SNHG16 (si-SNHG16), and si-NC were purchased from GeneChem (Shanghai, China). All transfections were carried out using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) in accordance with the manufacturer's instructions.
5
Establishing Bortezomib-Resistant Myeloma Cell Lines
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Cell culture. The human multiple myeloma cell lines, XG-7 and RPMI-8226, were purchased from the Cell Bank of the Chinese Academy of Sciences (CBCS; Shanghai, China). The establishment of bortezomib-resistant cell lines, XG-7/Bor and RPMI-8226/Bor, was performed using the gradient induction method by our study group. Drug-resistant cells were cultured in Invitrogen™ RPMI-1640 culture medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and 10 nM bortezomib (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) under culture conditions of 37˚C and 5% CO 2 . Cells showed semi-suspension growth, and cell passage was performed using the centrifugation method. In addition, 293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were used as the tool and virus package cell line, and they were cultured in high-glucose DMEM culture medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS under culture conditions of 37˚C and 5% CO 2 . These cells showed adherent growth and were passaged using trypsin digestion (Invitrogen; Thermo Fisher Scientific, Inc.) method when cell confluence reached 70%.
6
Myeloma Cell Line Cultivation and Treatments
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The human myeloma cell lines (HMCLs) RPMI8226 and MM.1S were obtained from the Cell Bank of the Chinese Academy of Sciences. NCI-H929, ARP-1, CAG, OPM2, and RPMI8226.BR and ARK cells were gifted by Dr. Qing Yi (Center for Hematologic Malignancy Research Institute, Houston Methodist, USA). All human cell lines have been authenticated using STR profiling and all experiments were performed with mycoplasma-free cells. BM samples from MM patients and peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained after informed consent was provided following approval by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University. HMCLs were cultured in RPMI-1640 medium (Corning Cellgro, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Gibco, USA) at 37 °C in a humidified atmosphere containing 5% CO2. Bortezomib, carfilzomib (CFZ), ixazomib (IXA), doxorubicin (ADM), melphalan (MEL), lenalidomide (LEN), rapamycin (Rapa), hydroxychloroquine sulfate (HCQ), Z-VAD-FMK, and NQDI-1 were purchased from Selleck Chemicals, LLC (Houston, TX, USA). The Bax inhibitor peptide V5 (Baxi) was obtained from MedChemExpress (New Jersey, USA).
7
Bone Marrow Analysis of Multiple Myeloma
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Bone marrow specimens were obtained from 15 newly diagnosed MM patients and 15 normal controls (NC) at Harbin Medical University Cancer Hospital, China, between January 2019 and December 2020. The National Comprehensive Cancer Network clinical practice guidelines were used to diagnose MM.
The MM cell line, RPMI 8226 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% (for RPMI 8226) fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and 1% penicillin–streptomycin at 37 ℃ in a humidified incubator containing 5% CO2.
The MM cell line, RPMI 8226 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% (for RPMI 8226) fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and 1% penicillin–streptomycin at 37 ℃ in a humidified incubator containing 5% CO2.
8
Cell Line Authentication and Modification
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HEK293T and K562 cells were purchased from the American Type Culture Collection. RPMI-8226 and MM.1S cells were purchased from the Cell Bank of the Chinese Academy of Sciences. All cell lines were authenticated by short tandem repeat profiling. RPMI-8226 and MM.1S cells stably expressing firefly luciferase (ffLuc) were established by lentiviral infection. K562 cells stably expressing BCMA and firefly luciferase (ffLuc) were generated by lentiviral infection. K562 cells stably expressing BCMA, PD-L1 and ffLuc were generated using a lentiviral vector containing a coexpression cassette for PD-L1 and ffLuc. All the stable cell lines were selected with puromycin. All cell lines were regularly tested to ensure that they were free of mycoplasma contamination.
9
Culturing Human Myeloma Cell Lines
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Human MM cell lines including U266 and RPMI8226 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and grown in RPMI1640 medium in the presence of 10% fetal bovine serum (FBS) and antibiotics at 37 degree in a 5% CO2 humidified environment.
10
Evaluating miR-342-3p and SNHG16 in MM
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Two human MM cell lines (RPMI-8226 and NCI-H929) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MM cell lines were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% penicillin–streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified atmosphere of 5% CO2. MiR-342-3p mimics, miR-342-3p inhibitor, miR-negative control (miR-NC), siRNAs to SNHG16 (si-SNHG16), and si-NC were designed and commercially constructed by Genechem (Shanghai, China). All transfections were conducted using Lipofectamine 2000 (Invitrogen). The sequences used were as follows: si-SNHG16, 5′-GGAACAUACUGCUAUCAUAGA-3ʹ; si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3ʹ; miR-342-3p mimics: 5′-UCUCACACAGAAAUCGCACCCGU-3ʹ; miR-NC: 5′-UUCUCCGAACGUGUCACGUTT-3ʹ; miR-342-3p inhibitor: 5′-ACGGGUGCGAUUUCUGUGUGAGA-3ʹ; NC inhibitor: 5′-CAGUACUUUUGUGUAGUACAA-3ʹ.
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