Plasmids containing shRNAs sequences for SPAK and OSR1 form the Sigma Mission library (PLKO) were purified from bacterial glycerol stocks (Table S2 ). Lentiviral particles (LVPs) were produced using a standardized protocol in the laboratory. It requires the transfection of HEK293T cells (ATCC® CRL-3216) with the transfer plasmid of interest, the packaging plasmid (psPAX2 Addgene), and the envelope vector (pMD.2 Addgene) in the presence of Lipofectamine 3000 (Thermo Scientific). The LVPs were then concentrated using Lenti-XTM Concentrator (Clontech) and resuspended in PBS for quantitative characterization by ELISA using the Lenti-X p24 Rapid Titer Kit (Takara). GBM cell 965 was transduced and selected using puromycin (Invitrogen). The knockdown efficiency was corroborated by obtaining lysates and performing WB using antibodies specific for OSR1 (Cell Signaling-#3729), SPAK (Cell Signaling-#2281).
Lenti xtm concentrator
Manufactured by Takara Bio
Sourced in France, United States
The Lenti-XTM Concentrator is a laboratory equipment designed for the concentration of lentiviral particles. It utilizes a proprietary filtration technology to efficiently concentrate lentiviral samples, enabling researchers to obtain higher viral titers from their cultures.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (8 mentions)
Hek293t cell, by American Type Culture Collection (3 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (3 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Lenti x p24 rapid titer kit, by Takara Bio (2 mentions)
Pspax2, by Thermo Fisher Scientific (2 mentions)
Pmd 2, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Saquinavir mesylate, by Merck Group (2 mentions)
Polyjet, by SignaGen (1 mentions)
Virapower lentiviral packaging mix, by Thermo Fisher Scientific (1 mentions)
Plenti cmv to sv40 small large t, by Addgene (1 mentions)
Lenti xtm qrt pcr titration kit, by Takara Bio (1 mentions)
Polybrene, by Merck Group (1 mentions)
Qpcr lentivirus titration kit, by Applied Biological Materials (1 mentions)
Lenticrispr v2, by Addgene (1 mentions)
Macs lineage depletion kit, by Miltenyi Biotec (1 mentions)
Quicktiter lentivirus quantitation kit, by Cell Biolabs (1 mentions)
Plenticmv gfp neo, by Addgene (1 mentions)
Pspax2, by Addgene (1 mentions)
21 protocols using lenti xtm concentrator
1
Lentiviral Knockdown of SPAK and OSR1 in GBM Cells
2
Lentivirus Production in HEK293T Cells
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To produce lentivirus, HEK293T cells were transfected with library DNA with PolyJet (SignaGen) according to the manufactory’s instructions. After 24 hours, the media was changed to complete DMEM (with 10% FBS and 1% P/S) containing 10 mM sodium butyrate (Bioshop). After another 24 hours incubation, the media was collected and centrifuged at 500 g at 4 °C for 10 min to pellet cell debris. The supernatant was collected and mixed with 1/3 volume of Lenti-XTM Concentrator (Clontech). Then the mixtures were incubated at 4 °C for overnight before being spun down at 1,500 g at 4 °C for 45 min. The virus pellets were resuspended with cold 1×PBS at 1/100 dilution and aliquoted before being frozen by liquid nitrogen.
3
Lentiviral Transduction of ESRRB in mESCs
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For lentivirus packing and processing, HEK293FT cells were transfected with 10 µg plasmid of pCDH-EF1-FLAG-ESRRBWT-T2A-copGFP (or pCDH-EF1-FLAG-ESRRBS25A-T2A-copGFP), 8 µg plasmid of PAX2, and 4 µg plasmid of MD.2G when the cells were grown to approximately 60% confluence in a 10-cm cell culture dish. The cell culture medium was filtered and collected after 72 h, followed by addition of Lenti-XTM Concentrator (Clontech, 631231) for lentivirus extraction based the manufacturer’s instructions. The resulting sample was resolved in PBS (pH 7.4) and added into the cell culture of R1 mESCs when the cells were grown to approximately 30% confluence. After incubation for 24 h, the medium was changed to fresh R1 cell culture medium. The cells were cultured for two passages, followed by flow cytometry sorting of mESCs expressing ESRRBWT or ESRRBS25A with equal GFP fluorescence intensity.
4
Generating Stable MIF-Expressing and Knockdown Cells
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Lentiviral vectors were used to generating the stable MIF-over expressing and shRNA MIF-knockdown cells as described by Funamizu et al. Briefly, Lentiviral MIF knock-in constructs (pLOC-MIF), and lentiviral MIF knock-down constructs (pGIPZ-shRNA 1 and shRNA2) were obtained from Open Biosystems (Rockford, IL). Lentiviral particles were produced by transfecting 239 T cells according to the manufacturer’s protocol. Viral supernatants was collected after 72 hours following transfection, and the particles were concentrated by using LentiXTM Concentrator over night at 4°C (Clontech, Mountain View, USA), the aliquots were stored at -80°C. The titers of the concentrated particles were measured before using. The PANC-1 and CAPAN-2 cells were (5× 105cells/well)were seeded in 6-well culture plates, maintained in DMEM medium with 10% FBS for 24 hours before infection. For screening, blasticid in (10 μg/mL) for MIF Knock-in cells and puromycine (10 μg/mL) for MIF knock-down cells were added to the medium 72 hours after infection. The medium was replaced every 2 days for 2–3 weeks.
5
Establishment of Immortalized Keratinocytes
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Primary keratinocytes obtained from 2-day-old K14Cre.Ptpn2w/w (TC-PTP/WT) or K14Cre.Ptpn2fl/fl (TC-PTP/KO) neonates were cultured in keratinocyte growth medium (PromoCell, #C-20211, C-39011) containing 1% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO2 according to the previously described method48 (link). Mouse immortalized keratinocyte cell lines were established by using pLenti CMV/TO SV40 small + large T (Addgene, #22298). Viral packaging using 293T cells and titration of viruses were performed by using ViraPowerTM Lentiviral Packaging Mix (Invitrogen, K497500). The packed virus was concentrated by Lenti-XTM Concentrator (Clontech, 631231). For establishing stable immortalized keratinocyte cell lines, primary keratinocyte cells were infected with lentivirus containing the pLenti SV40 small + large T-antigen-expressing vector. After transduction, cells were cultured with keratinocyte growth medium for over ten passages to select for the clones capable of growing indefinitely.
6
SARS-CoV-2 Pseudotyped Virus Production
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The following plasmids were obtained from Addgene including pcDNA3.1-SARS2-Spike mammalian expression plasmid (#145032), NL4-3 mCherry Luciferase dual reporter vector (#44965), and the control env expression vector, VSV-G (pMD2.G) (#12259). The SARS-CoV-2 G614 mutant expression vector was generated as recently reported (Zhang et al., 2020a ). The SARS-CoV-2 pseudotyped virus was produced by transfecting 293T cells with 10 μg of NL4-3 mCherry Luciferase and 10 μg env expression vector: either VSV-G (pMD2.G) or pcDNA3.1-SARS2 Spike using polyethylenimine (PEI) (25 kDa, 1 μg/μL). The virus supernatant was collected at 48 h after transfection and concentrated at 1:100 ratio using Lenti-XTM Concentrator (TaKaRa/Clontech, 631231). Viral titer was determined using Lenti-XTM qRT-PCR Titration Kit (TaKaRa/Clontech, 631235).
7
Lentiviral Transduction of bEnd.3 Cells
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The lentiviral particles were produced in HEK293T cells (ATCC, CRL-3216) using PEI transfection method. The lentivirus was purified by Lenti-XTM Concentrator (Clontech, 631231) and tittered by qPCR Lentivirus Titration Kit (Applied Biological Materials Inc., LV900-S). bEnd.3 cells were plated in 6-well plates and allowed to adhere overnight. Virus was added at the indicated titer in the presence of polybrene (2μg/mL, Sigma, H9268) and the cells were incubated for 24 h. After 24 h, the media was changed to fresh media with no virus and the cells were incubated for additional 4–5 days, then subjected to the subsequent experiments (e.g., Western-blotting and in vitro-uptake experiments).
8
Lentiviral CRISPR Transduction in U2OS Cells
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Single guide RNA (sgRNA) sequences (Table S1 ) were cloned into lentiCRISPRv2 (Addgene, #52961) by using NEBuilder HiFi DNA assembly reagents (New England Biolabs). For lentivirus production, 60-70% confluent HEK293T cells were transfected with sgRNA containing lentiCRISPRv2, pMDLg/pRRE (Addgene, #12251), pRSV-Rev (Addgene, #12253) and VSV.G (Addgene, #14888) plasmids by using Lipofectamine 2000 (Invitrogen). 72 h after transfection, media was collected and centrifuged at 3,000 rpm for 10 min at 4°C to pellet the cell debris. The supernatant was filtered through a 0.45 μm low-protein-binding membrane. Viral particles were further concentrated by adding Lenti-XTM concentrator (Clontech) to the supernatant at a 1:3 ratio. The mixture was incubated for 2 h at 4°C and centrifuged at 1,500 g for 45 min at 4°C. After removing the supernatant, the pellet containing lentiviruses was resuspended in 1 mL DMEM, aliquoted, and stored at −80°C.
For lentivirus transduction, 2 × 105 U2OS cells were seeded in each well of 24-well plates. One aliquot of viral particles was added to each well and mixed. After 48 h, fresh media containing 1 μg/mL puromycin were added for selection. After two rounds of 48-h selection, puromycin-resistant cells were harvested and expanded for downstream analyses.
For lentivirus transduction, 2 × 105 U2OS cells were seeded in each well of 24-well plates. One aliquot of viral particles was added to each well and mixed. After 48 h, fresh media containing 1 μg/mL puromycin were added for selection. After two rounds of 48-h selection, puromycin-resistant cells were harvested and expanded for downstream analyses.
9
Gamma Radiation Effects on Bone Marrow Transplants
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All mouse strains were bred from C57/BL6 mice to ensure consistency. Bone marrow transplants were performed on 5 animals of equal age, weight and sex ratios (1:1). National Institutes of Health guidelines for the care and use of laboratory animals and the recommendations of the ** laboratory animal ethics committee were followed throughout all animal research. The MACS lineage depletion kit (Miltenyi Biotec GmbH, Germany) was used to separate bone marrow cells from the femur and tibia bone marrow of an 8-week-old C57/BL6 mice. The procedure was carried out in accordance with the guidelines provided by the manufacturer. Using a Lenti-XTM concentrator (Clontech, Canada), we infected the recovered stem cells twice with either a highly purified form of the mice MALAT1 shRNA lentivirus or a scramble lentivirus.25 (link) To test the effects of a high dosage of gamma radiation, 9.6 Gy was administered to C57/BL6 mice that were around 8 weeks old. One million infected cells were injected retro-orbitally into the irradiated mice. Platelets were extracted from recipient mice 4–6 weeks after transplantation.
10
Lentiviral Knockdown of SPAK and OSR1 in GBM Cells
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