Phoenix packaging cells (#CRL-3214; ATCC) were transfected with 20 µg of the plasmid pCL-Eco (#25099; Addgene) and 16 µg of pMXs-PPT1, pMXs-OVA (cloned from Addgene #25099), or empty vector pMXs with 90 µl transfecting reagent polyethylenimine (23966-1; Polysciences) at 1 mg/ml in a 10-cm culture dish. After 48 h, the supernatant containing retrovirus was harvested and filtered. For BMDC retroviral transduction, on days 1 and 2, 2 ml retroviral supernatant was added, and cells were spin-infected (2,500 rpm) for 90 min at room temperature. On day 3, fresh medium was added to the cells, and they were cultured for two more days. All retroviral transduction rate was >70% as measured by GFP with FACS.
Phoenix packaging cells
Manufactured by American Type Culture Collection
Phoenix packaging cells are a type of laboratory equipment used in cell culture and molecular biology research. They serve as a tool for the production of viral particles. The core function of Phoenix packaging cells is to provide a standardized and reproducible system for the generation of viral vectors, which can be used in various experimental applications.
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Lab products found in correlation
Lipofectamine ltx, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (2 mentions)
Pcl eco, by Addgene (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Bovine pituitary extract, by Atlanta Biologicals (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
5 protocols using phoenix packaging cells
1
Retroviral Transduction of Murine Dendritic Cells
2
Retroviral Transduction of FIPs
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Retrovirus was packaged in Phoenix cells as previously described 17 (link). Briefly, Phoenix packaging cells (ATCC CRL-3213) were co-transfected with 10 μg of the pMSCV overexpression plasmids (pMSCV-Zfp423-WT, pMSCV-Zfp423-ΔNID or empty vector) and 5 μg gag-pol and 5 μg VSV-g plasmids using Lipofectamine LTX (ThermoFisher Scientific, #15338100). Subsequently, FIPs were transduced with diluted virus (1:10 ratio) in ITS media containing 8 μg/ml polybrene (Sigma, #TR-1003) for 24 hours. Following transduction, cells were maintained in ITS media for ≥ 48 hours before beginning treatments.
3
Cultivation and Transduction of NHPrE1 Cells
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NHPrE1 cells [18 (link)] were maintained in DMEM (Gibco) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA), 1% penicillin/streptomycin (Gibco), 10 ng/ml epidermal growth factor (Sigma– Aldrich, St. Louis, MO), 1% insulin-transferrin-selenium (ITS) (Gibco), and 0.4% bovine pituitary extract (Atlanta Biologicals). NHPrE1 cells were transfected with EGFP-expressing plasmid; GFP-positive NHPrE1 cells were selected by cell sorting. To establish AR-expressing NHPrE1/GFP cells, CMV promoter-driven LNCX or LNCX-AR retroviral vector-based plasmids were transfected into Phoenix packaging cells (ATCC, Manassas, VA). Twenty-four hours later, culture media were collected and used to infect NHPrE1/GFP cells. The infection procedure was repeated twice. The transduced cells, stably expressing AR, were selected by culturing them in the presence of G418 (Sigma, St. Louis, MO, USA). G418 resistant cell populations were used in this study. American Type Culture Collection (ATCC) has authenticated NHPrE1 cells and no contamination from other type of cells was found. PC3/AR [21 (link)] and LNCaP (ATCC) cells were cultured in RPMI1640 supplemented with10% serum.
4
Engineered Melanoma Cell Lines for Vaccine Evaluation
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pMFG-OVA257–264-Cerulean, pMFG-GP61–80-Cerulean, and pMFG-OVA257–264-GP61–80-Ceruelan plasmids were constructed by inserting annealed oligonucleotides encoding triple SIINFEKL-AAY repeats, GLKGPDIYKGVYQFKSVEFD, or (SIINFEKL-AAY)3-P2A-GLKGPDIYKGVYQFKSVEFD, respectively, into the NcoI-linearized pMFG-Cerulean vector, as previously described [45 (link)]. Restriction enzymes were purchased from New England Biolabs. All constructs were verified by sequence analysis. Phoenix packaging cells (ATCC) were transfected with pMFG constructs; supernatants were used to transduce B16-F10 mouse melanoma tumor cell line to generate B16-F10 OVA257–264-Cerulean, B16-F10GP61–80-Cerulean and B16-F10 OVA257–264-GP61–80-Cerulean, respectively [45 (link)]. Transduced bulk cell lines were sorted for similar Cerulean expression levels.
5
Retroviral Transduction of FIPs
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