HEK293T cells were transfected with plasmids using polyethylenimine (PEI) (Polysciences, #24765). For retrovirus and lentivirus production, GP2–293 packaging cells (Clontech) or pCMV-DeltaR8.2 were used. After seventy-two hours of transfection, the virus-containing media was collected and used to spin-infect the cells at 1,800 rpm for 40 min. Cells were then incubated with the viral supernatant overnight. For siRNA transfection, the Neon transfection system (Thermo Fisher Scientific, #MPK5000) was used.
Gp2 293 packaging cells
Manufactured by Takara Bio
Sourced in United States
The GP2-293 packaging cells are a stable, adherent cell line derived from human embryonic kidney 293 cells. These cells are designed to produce high-titer, replication-incompetent retroviral particles when co-transfected with a retroviral vector and an envelope expression plasmid.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (4 mentions)
Puromycin, by Merck Group (3 mentions)
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
Facsaria fusion sorter, by BD (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pgfp 5 rs vector, by OriGene (2 mentions)
Retronectin, by Takara Bio (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Ba f3, by RIKEN BioResource Center (2 mentions)
Rpmi 1640, by Fujifilm (2 mentions)
Pmscv puro retroviral vector, by Takara Bio (1 mentions)
Pgl3 basic, by Promega (1 mentions)
Pvsv g, by Takara Bio (1 mentions)
Pvsv g vector, by Takara Bio (1 mentions)
Cts optmizer, by Thermo Fisher Scientific (1 mentions)
Cts immune cell serum replacement, by Thermo Fisher Scientific (1 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
26 protocols using gp2 293 packaging cells
1
Efficient Virus Production and Delivery
2
Retroviral Transduction of Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For retroviral transduction of RAW macrophages, VSV-G-pseudotyped retrovirus was made in GP2–293 packaging cells (Clontech). GP2–293 cells were transfected with retroviral vectors and pVSV-G using Lipofectamine LTX reagent. 24h post-transfection, cells were incubated at 32°C. 48h post-transfection viral supernatant (with polybrene at final 5μg/ml) was used to infect target cells overnight at 32°C and protein expression was checked 48h later. Target cells were sorted on a BD FACSAria Fusion Sorter to match expression or drug-selected with Puromycin, starting 48h after transduction. Efficiency of drug selection was verified by equal mCherry expression of target cells.
For retroviral transduction of bone marrow derived macrophages, bone marrow was harvested and cultured in M-CSF containing RPMI for two days. Progenitor cells were transduced with viral supernatant (produced as above) on two successive days by spinfection for 90min at 32°C. 48h after the second transduction cells were put on Puromycin selection and cultured in M-CSF containing RPMI media until harvested on day 8.
For retroviral transduction of bone marrow derived macrophages, bone marrow was harvested and cultured in M-CSF containing RPMI for two days. Progenitor cells were transduced with viral supernatant (produced as above) on two successive days by spinfection for 90min at 32°C. 48h after the second transduction cells were put on Puromycin selection and cultured in M-CSF containing RPMI media until harvested on day 8.
3
Retroviral Transduction of Macrophages
4
Generating Luciferase-Expressing Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Firefly (Photinus pyralis) luciferase cDNA from pGL3 basic (Promega, Madison, WI, USA) was inserted into the pMSCV puro retro-viral vector (Clontech, Palo Alto, CA, USA), generating pMSCV-luciferase. GP2-293 packaging cells (Clontech) were co-transfected with pMSCV-luciferase and pVSV-G (Clontech), a plasmid encoding the viral envelope glycoprotein (VSV-G) of vesicular stomatitis virus, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Supernatants from transfected GP2-293 were induced with approximately 50% confluent cells in the presence of polybrene (8 μg/ml final concentration; Sigma-Aldrich). The transduced cells were propagated in medium containing puromycin (Sigma-Aldrich) at 15 μg/ml and the generated cells were named luc-A549, luc-A549-FL, luc-H441, and luc-H441-FL. Cell morphology and growth characteristics of the cells were unchanged after transduction of the luciferase gene (S1 Fig ). Before the in vivo experiment, the luciferase activity of each cell type was measured in vitro and found to be similar.
5
Retroviral Expression Vectors for NSC-34 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To produce infecting retroviral expressing vectors, GP2-293 packaging cells (Clontech) were co-transfected with the above described TERC or alTERC constructs and with pVSV-G (Clontech). The medium containing the retroviral particles was used for the infection of NSC-34 cells. Cells containing the TERC or alTERC expressing vectors were selected by growing the infected NSC-34 cells with G418.
6
Generating Girdin knockdown 293FT cell line
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate stable Girdin knockdown 293FT cells, 24 μg of either control or Girdin shRNA and 4 μg of vesicular stomatitis virus G protein (pVSV-G) vector (Clontech) were cotransfected into GP2-293 packaging cells (Clontech Laboratories). Twenty-four hours after the transfection, the medium was replaced with 5 mL of fresh DMEM containing 10% FBS and the cells were further cultured at 32°C to facilitate virus production. HeLa cells were infected with virus-containing supernatants harvested 48 h post-transfection, followed by selection with 2 μg/mL puromycin.
7
siRNA-mediated Knockdown of IGF2BP3 in S2-013 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exponentially growing GP2-293 packaging cells (Clontech, Mountain View, CA) were transiently infected with pGFP-V-RS vectors (OriGene Technologies, Rockville, MD) to generate replication-deficient lentivirus that carried a small interfering RNA (siRNA) expression cassette targeting either a scrambled negative control (TR30013), or IGF2BP3 mRNA (TG312221). Upon transient transfection of the plasmids into the packaging cell line, replication-deficient viruses were obtained and used to infect S2-013 cells; infected S2-013 cells were transferred to flasks 48 h after infection and then grown in DMEM containing 0.5 μg/mL puromycin (Sigma-Aldrich) for 7 days to establish S2-013 cells that stably expressed the appropriate siRNA that targeted IGF2BP3 mRNA. For each experiment, these cells were cultivated until they reached confluence and then for an additional 10 days; medium was refreshed every second day during cell cultivation. Cells were used only when suppression of IGF2BP3 had been validated via western blot analysis.
8
Viral Transduction of HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were transfected with plasmids using polyethylenimine (PEI) (Polysciences, #24765). For retrovirus and lentivirus production, GP2-293 packaging cells (Clontech) or pCMV-DeltaR8.2 were used. After 72 h of transfection, the virus-containing media was collected and used to spin-infect the cells at 1800 rpm for 40 min. Cells were then incubated with the viral supernatant overnight. For siRNA transfection, the Neon transfection system (Thermo Fisher Scientific, #MPK5000) was used.
9
Retrovirus Production in GP2-293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5 x 106 GP2-293 packaging cells (Clontech) were plated in a 10 cm dish containing D10 medium (DMEM supplemented with 10% FBS, penicillin, streptomycin and glutamine). 24 hours after plating, 25 μg of the retroviral expression vector (pLZRS-human codon-optimized Bcl6-P2A-human codon-optimized BclxL-IRES-GFP) and 5 μg of the envelope vector (p10A1; Clontech) were transfected into GP2-293 cells using Lipofectamine 3000 (ThermoFisher) according to the manufacturer’s instructions. After 24 hours, the cells were supplemented with fresh D10 media. Retroviral supernatant was harvested 48 hours post-transfection, replaced with fresh D10 and again harvested 72 hours post-transfection.
10
Generation of Anti-c-Met CAR T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human PBMCs were collected from healthy volunteers, under the institutional approval of Yamaguchi University. The constructs of anti‐c‐met single‐chain variable fragment (scFv) were generated based on previous reports.41 The scFv was fused with the transmembrane domain of human CD8α, and cytoplasmic regions of human CD28, 4‐1BB (CD137), and CD3ζ, to construct a 3rd generation CAR, which was then cloned into pMSGV1 to generate retroviral vectors.41 , 42 Transduction of the CAR‐expressing retroviral vectors into human T cells was conducted as previously described,41 , 43 with some modifications. Briefly, GP2‐293 packaging cells (Clontech, Mountain View, CA) were transfected with the CAR‐expressing plasmid together with p‐Ampho retrovirus packaging plasmid (Clontech) using Lipofectamine® Reagent (Thermo Fisher Scientific). Culture supernatants containing retroviral vectors were harvested and used for gene transduction. Activated healthy donor‐derived PBMCs were infected with viral supernatants in the presence of RetroNectin® (TaKaRa Bio, Kusatsu, Japan). Cells were incubated with OpTmizer (Gibco) supplemented with OpTmizer CTS, CTS Immune Cell serum replacement, l ‐glutamine (Gibco), penicillin‐streptomycin sulfate, and amphotericin B for 5 d in the presence of IL‐2. Transduction efficiency of CAR was assessed using flow cytometry.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!