HEK-293T cells were seeded at a density of 1.0 × 10^6 cells per plate in a 6 cm tissue culture dish overnight with low antibiotic growth media (DMEM +10% FBS). Cells were incubated until 70% confluence was achieved. A mixture of three transfection plasmids were produced by combining 2 μg pMDG.2 (Addgene #12259) (Addgene, Watertown, Massachusetts, USA); 4 μg of pCMV delta R8.2 (Addgene #12263) and 5 μg of vector (pCDH-CMV-MCS-EF1-GFP empty, pCDH-CMV-MCS-EF1-GFP-C/EBPδ - C/EBPδ-OE). As per manufacturer's protocol, GenJet DNA In-vitro Transfection reagent (Ver. II) (SignaGen Laboratories, Rockville, MD, USA) was used as the transfection reagent. Reagents were added to each plate drop-wise and was incubated overnight (16 h, 37 °C, 5% CO2). Next day, media was subsequently removed and replaced with high BSA growth media and again incubated for 24 h. Supernatant was then collected and passed through a 0.45 μm filter and snap frozen and stored at −80 °C until required for use. A second round of collection was performed 72 h after virus transfection and was also filtered and stored at −80 °C.
Pcmv delta r8
Manufactured by Addgene
Sourced in United States, France
The PCMV delta R8.2 is a lentiviral packaging plasmid used for the production of lentiviral particles. It contains the necessary viral genes for packaging lentiviral particles but lacks the viral genome, making it a useful tool for safe and efficient lentiviral gene delivery.
Automatically generated - may contain errors
Lab products found in correlation
Pcmv vsv g, by Addgene (15 mentions)
Pmd2 g, by Addgene (9 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Pcmv vsv g vector, by Addgene (3 mentions)
Plko 1 lentiviral vector, by Merck Group (2 mentions)
Tmem38b, by Merck Group (2 mentions)
Polybrene, by Merck Group (2 mentions)
Pmd2 g vsvg, by Addgene (2 mentions)
Opti mem, by Addgene (2 mentions)
Amicon ultra 15 centrifugal ultrafilters, by Merck Group (2 mentions)
Steriflip, by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Gw1 phred, by Addgene (2 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (2 mentions)
Shlcn2 1 trcn0000060289, by Merck Group (2 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Blasticidin, by Thermo Fisher Scientific (2 mentions)
Pmdg 2, by Addgene (1 mentions)
38 protocols using pcmv delta r8
1
Production of Lentiviral Particles
2
Lentiviral construction and transduction of LifeAct fusion proteins
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The plasmid encoding LifeAct-EGFP was a kind gift from Professor Wedlich-Söldner54 (link). To construct LifeAct-mCherry, the EGFP tag was excised by double digestion with the AgeI and BsrgI restriction enzymes, and the mCherry was ligated using the same sites. LifeAct-EGFP and LifeAct-mCherry were subsequently sub-cloned into the lentiviral vector (pFUGW). The plasmids containing the LifeAct-mCherry or LifeAct-EGFP fusion fragments were double-digested with XbaI and NheI, and subsequently the LifeAct-EGFP or LifeAct-mCherry fusions were gel-purified and sub-cloned into the lentiviral vector pFUGW using XbaI cloning sites. Recombinant lentiviruses were produced by triple transfection of the packaging 293T cells with the lenti FUGW carrying the gene of interest, packaging vectors (pCMV delta R8.2) and the envelope vector (pMD2.G) carrying the VSV glycoprotein (purchased from Addgene, Cambridge, MA 02139, USA). The virus was concentrated by ultracentrifugation at 50,000 × g for 90 min at 4 °C, and the pellet was stored at −80 °C until use.
3
Lentiviral Transduction of Mammalian Cells
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The pCMVdeltaR8.2 and pCMV-VSV-G vectors were purchased from Addgene (Cambridge, MA, USA). The pLV-SV40-puro lentiviral vector was obtained from Dr. Peter Chumakov, Cleveland Clinic (Cleveland, OH, USA). Human KLF9 or TXNRD2-expressing lentiviral vectors were described previously [10]. XBP1s expressing plasmid was purchased from Genecopoeia (Rockville, MD). The pLKO-1 lentiviral vectors containing control shRNA and shRNAs for KLF9, ITPR1, TMEM38b, XBP1, ATF4, and ATF6 were purchased from Sigma-Aldrich. The lentiviral infection protocol has been described previously (Zucker et al., 2014 (link)). Equivalent amounts of control virus were used in all experiments.
4
Lentiviral Transduction and Monoclonal Selection
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Lentiviral production and transduction were performed as described previously [19 (link)]. Briefly, HEK293T cells were co-transfected with pMD-VSVG, pCMV-delta R8.2 (Addgene, Beijing, China), and a lentiviral vector loaded with the target gene using polybrene [20 (link)]. Virus-containing medium was harvested at 48 h after transfection. FRT cells were infected with 1 mL of virus supernatant and 4 μg/mL of polybrene (Sigma-Aldrich, Shanghai, China) and then further cultured for 48 h before screening for overexpression monoclonal clones expressing resistance through the use of 0.5 μg/mL of puromycin (YFP-H148Q/I152L high-expression lentiviral vector) or 50 μg/mL of blasticidin (Ano1 high-expression lentiviral vector). In this experiment, the monoclonal overexpression vector of Ano1 was preferentially constructed, and the Ano1 monoclonal cells were expanded and cultured. They were then screened with YFP-H148Q/I152L high-expression lentivirus after infection to obtain the final co-transformed Ano1 and YFP-H148Q/I152L FRT cell line.
5
SHIP1 Knockdown in MEC-1 CLL Cells
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shRNA SHIP1 knockdown1 (KD1) and shRNA SHIP1 knockdown2 (KD2) as well as scramble (scr) were purchased from Sigma Aldrich and subcloned into pLKO.1. For the generation of SHIP1 KD MEC-1 cell lines, MEC-1 Eco and MEC-1 Eco/Luc cells were transduced with scramble or 2 constructs of SHIP1-targeting shRNA (KD1, KD2) concentrated lentivirus (pCMV delta R8.2: Addgene #12263, RRID: Addgene_12263), Phit123: kindly provided by Markus Müschen). 5 days after spin infection, Puromycin selection was initiated with 1 μg/ml for 3 days. Cell viability before and after Puromycin selection was assessed by flow cytometric DAPI exclusion and by total cell count. SHIP1 knockdown was confirmed by western blotting. 1 × 106 shSHIP1 KD1, KD2 MEC-1 cells, and shScramble control-containing MEC-1 cells were transplanted into NSG mice and CLL engraftment and progression was followed by bioimaging as described above. Bone marrow and spinal cord cells were isolated from sacrificed mice and MEC-1 (GFP+, CD19+) cells were FACS sorted and SHIP1 expression levels were analyzed by qPCR.
6
Retroviral and Lentiviral Transduction of Mouse Cells
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Ecotropic retroviruses were prepared by co-transfecting HEK 293 T/17 cells (ATCC® CRL-11268) in a 10-cm plate with 10 μg of packaging pCL-ECO (Addgene plasmid # 12371) and 10 μg retroviral based vectors by using Lipofectamine 2000 Transfection Reagent (Invirtogen) according to manufacturer’s protocol. For lentiviral infection, transfer vectors were co-transfected into HEK 293 T/17 cells with packaging pCMV delta R8.2 (Addgene plasmid # 12263) and envelope pVSV-G (Clontech, PT3343-5) vectors. Viruses were harvested 40 h and 64 h after the transfection and filtered through a 0.45-µm PES filter (Millipore). For viral infections, 5 × 105 mouse bone marrow cells or 32Dcl3 cells were resuspended in 1 ml of virus-containing medium with 6 μg/ml polybrene (Sigma). GFP, RFP or mCherryFP -positive cells were sorted 48 h after the initial infection.
7
Lentiviral Overexpression of Neuronal Factors
8
Generation of Thy1.1-expressing Lentivirus
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The pLentiGuide-Puro plasmid was a gift from Feng Zhang (Addgene plasmid # 52963) [28 (link)]. The Puromycin resistance cassette was replaced with Thy1.1, including the insertion of a silent mutation to eliminate a BsmBI site in Thy1.1, by the Custom Cloning Core of the Emory Integrated Genomics Core. sgRNAs were cloned into the BsmBI site according to previously described protocols [48 (link)]. LentiX-293T cells (Takara; 632180) were cultured with high glucose (4.5 g/L) DMEM supplemented with 10% heat-inactivated FBS, 2 mM l -glutamine, 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin. For lentiviral production, LentiX-293T cells were seeded at 5 million/10 ml in a 10 cm dish for 20–22 h. The pCMV delta R8.2 (Addgene; 12263), pCMV-VSV-G (Addgene; 8454), pLentiGuide-Thy1.1 plasmid were mixed at a 4:1:5.4 M ratio in Opti-MEM I medium (Gibco; 31985070) to make a 500 μl suspension. 50 μl TransIT-293 reagent (Mirus; MIR 2700) was then added, mixed, and allowed to sit at room temperature for 30 min. The suspension was added dropwise into each plate and mixed by gentle shaking of the plate. Following a 16–18 h incubation, the media was replaced with 15 ml DMEM growth medium supplemented with 1% BSA. Lentiviruses were harvested 24 and 48 h later and purified by ultracentrifugation using a SW28 and SW41 rotor (Beckman; 342207 and 331362, respectively).
9
Rapid Insulin Secretion Monitoring
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We used the proinsulin-NanoLuc plasmid (David Altshuler, Addgene plasmid #62057, proinsulin-NanoLuc in pLX304) to provide a low-cost, scalable, and rapid method to detect insulin secretion. The gene encoding NanoLuc was cloned into the C-peptide portion of mouse proinsulin such that cleavage within insulin vesicles by pH-sensitive prohormone convertase results in the co-secretion of NanoLuc with endogenous insulin in a stimulus-dependent manner (Burns et al., 2015 (link)). The pLX304 lentivirus packaging plasmid containing the proinsulin-NanoLuc construct was transfected into HEK293T (ATCC CRL-11268) cells with pCMV-VSVG (envelope vector, Addgene plasmid #8454) and pCMV delta R8.2 (packaging vector, Addgene plasmid #12263). Supernatant containing lentivirus particles was harvested 48 hr after transfection. Beta-TC-6 and Bmal1-/- Beta-TC-6 cells were infected with insulin-NanoLuc lentivirus, and stably expressing cells were selected by treating with puromycin (2 µg/ml, 2 days).
10
Lentiviral Vector Production in HEK293T Cells
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HEK293T cells were maintained in DMEM supplemented with 10% FBS. Cells were seeded into a 15 cm dish such that cells were 60%–70% confluent. 36 μl Lipofectamine 2000 (Life Technologies) was added to 1.5 ml of Opti-Mem (Life Technologies) in one tube and 3 μg of pMD2.G (Addgene #12259), 12 μg of pCMV delta R8.2 (Addgene #12263), and 9 μg of each individual vector was added to 1.5 ml of Opti-Mem in another tube. After 5 min of incubation, tubes were mixed and incubated for 30 min. The solution was then added dropwise to the 15 cm dish. Viral-containing supernatant was harvested after 48 and 72 h, filtered with Steriflip (Millipore), concentrated to 1 ml using Amicon Ultra-15 centrifugal ultrafilters with a 100 000 NMWL cutoff (Millipore), and frozen at −80 °C.
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