HEK293LTV and HEK293Ad cells (Cell Biolabs) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Gaithersburg MD) supplemented with 10% Fetal Bovine Serum (Wisent) 1× Penicillin/Streptomycin (Thermofisher, Rockville, MD), 1× Glutamax (Thermofisher). Cells co-transfected with pAAV2/8 (PennVectorCore), pHelper and pAAV-MCSCII or pAAV-MCSGFP (Cell Biolabs) at a molar ratio of 1:1:1 by Calcium Phosphate (Takara, Ann Arbour, MI) according to manufacturer’s directions. Vector purification and titration were performed as previously described23 (link),24 (link). Ad5 was produced in HEK293Ad cells. Plasmids pacAd5 CMV K-N: CII and pacAd5 9.2–100 (Cell Biolabs) were co-transfected at a 1:1 molar ratio with Calcium Phosphate (Takara) according to manufacturer’s directions. The virus was released by freeze–thaw and subsequently amplified in HEK293Ad cells and purified as previously described25 .
Calcium phosphate
Manufactured by Takara Bio
Sourced in United States
Calcium phosphate is a class of inorganic compounds used in various laboratory applications. It serves as a general-purpose reagent, with the core function of serving as a precipitating agent and transfection enhancer.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
C57bl 6 mice, by R&D Systems (3 mentions)
M csf, by R&D Systems (3 mentions)
Lipofectamine and plus reagent, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Wild type c57bl 6 mice, by Samtako (2 mentions)
Dulbecco modified eagle medium (dmem), by R&D Systems (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Pcmv6 entry plasmid, by OriGene (2 mentions)
Myc ddk tagged human rpl5, by OriGene (2 mentions)
Mrpl18, by OriGene (2 mentions)
Linear polyethylenimine pei, by Polysciences (2 mentions)
Lenti x concentrator, by Takara Bio (2 mentions)
Mouse anti flag m2 antibody conjugated beads, by Merck Group (2 mentions)
Peg it virus precipitation solution, by System Biosciences (2 mentions)
23 protocols using calcium phosphate
1
Protocol for Adenovirus and AAV Production
2
GST-pulldown Assay for Protein Interactions
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For GST-pulldown assays using HEK293T cell lysates, cells were transfected with the GFP construct-containing DNA using calcium phosphate (Takara Bio) transfection. After 24 h, cells were harvested, washed with PBS, and lysed in immunoprecipitation lysis buffer solution (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 2 mM EDTA; 1% Triton X ‐100; and protease and phosphatase inhibitors), and the supernatants were isolated after centrifugation. Cell lysates were incubated with purified GST-mATG8 protein and glutathione-agarose beads overnight at 4 °C. The next day, they were washed 3–5 times with immunoprecipitation lysis buffer solution at 4 °C. Proteins were separated by SDS–PAGE and analyzed by Western blot and Coomassie blue staining.
3
Lentivirus Production via Calcium Phosphate or Lipofectamine
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Lentivirus was produced using Calcium phosphate (Takara Bio, Mountain View, CA) or Lipofectamine (Invitrogen, Carlsbad, CA) as described [15 (link)].
4
GST Pulldown Assay for Protein Interactions
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For the GST pulldown assays with HEK293T cell lysates, cells were transfected with the DNA encoding the indicated 3xFLAG or GFP constructs using calcium phosphate (Takara Bio) transfection. After transfection, cells were harvested, washed with PBS, lysed in immunoprecipitation lysis buffer solution (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% TX-100, and protease and phosphatase inhibitors) and removed by centrifugation. Cell lysates were incubated with purified GST-mATG8 proteins with glutathione-agarose beads overnight at 4°C. The following day they were washed with an immunoprecipitation lysis buffer solution at 4°C; this step was repeated three to five times. Proteins were then resolved using SDS–PAGE and analyzed using Western blot analysis.
5
Transfection and Culturing of HEK293T and MEF Cells
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HEK293T cells and established MEF cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin/streptomycin in a humidified atmosphere with 5% (v/v) CO2 at 37°C. Cells were seeded in a 6-well cell culture plate (Catalog #: 30006; SPL, Gyeonggi, Korea) or a sticky-slide eight-well system (Catalog #: 80828; Ibidi, Martinsried, Germany) to obtain 40%-60% confluent cells on the day of imaging. Cells were transfected with the indicated DNA constructs using calcium phosphate (Catalog #: 631312; Takara Bio inc., Shiga, Japan) or Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) 24-26 h before analysis. The relative amount of each construct was empirically calculated according to the relative expression of each construct combination.
6
Lentivirus Production Using Calcium Phosphate or Lipofectamine
7
Lentivirus Production and Concentration
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Lentivirus was produced in HEK‐293T cells co‐transfected with the pHIV7 lentiviral vector and the packaging vectors pCHGP‐2, pCMV‐rev2 and pCMV‐VSV‐G, (Addgene) using calcium phosphate (Clontech). Medium was changed 16 h after transfection, and the cells were incubated for another 36–48 h. Lentiviral supernatants were collected, centrifuged to remove cellular debris for 15 min at 2217 g and concentrated for 2 h at 55126 g. The lentiviral pellet was resuspended in TBS‐5 buffer, snap‐frozen on dry ice and stored at −80°C.
8
Isolation and Culture of Mouse Bone Marrow-Derived Macrophages
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Wild-type C57BL/6 mice were purchased from Samtako Bio Korea (Gyeonggi-do, Korea). Primary bone marrow–derived macrophages (BMDMs) were isolated from six-week-old C57BL/6 mice and cultured in DMEM for 3–5 days in the presence of M-CSF (R&D Systems, 416-ML), as described previously [32 (link)]. HEK293T (ATCC-11268; American Type Culture Collection) or THP-1 (ATCC-TIB-202) cells were maintained in DMEM or RPMI1640 (Gibco, NY, USA) containing 10% FBS (Gibco, NY, USA), sodium pyruvate, nonessential amino acids, penicillin G (100 IU/mL), and streptomycin (100 μg/mL). Transient transfections were performed using calcium phosphate (Clontech, Mountain View, CA, USA) in 293T, according to the manufacturer’s instructions. THP-1 stable cell lines were generated by transfections were performed using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) and then a standard selection protocol with 400–800 μg/mL of G418.
9
Bone Marrow-Derived Macrophage Isolation and Manipulation
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Wild‐type C57BL/6 mice were purchased from Samtako Bio Korea (Gyeonggi‐do, Korea). Primary bone marrow‐derived macrophages (BMDMs) were isolated from C57BL/6 mice and cultured in DMEM for 3–5 days in the presence of M‐CSF (R&D Systems, 416‐ML), as described previously (Koh et al, 2017 ). BMDMs of TLR2−/−, TLR4−/−, MyD88−/−, TRIF−/−, IRAK1−/−, TRAF6−/−, and TBK1−/− in C57BL/6 mice were a generous gift from Dr. Chul‐Ho Lee (Laboratory Animal Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea). HEK293T (ATCC‐11268; American Type Culture Collection) and RAW264.7 (ATCC TIB‐71) in DMEM (Gibco) containing 10% FBS (Gibco), sodium pyruvate, nonessential amino acids, penicillin G (100 IU/ml), and streptomycin (100 μg/ml). Human monocytic THP‐1 (ATCC TIB‐202) cells were grown in RPMI 1640/glutamax supplemented with 10% FBS and treated with 20 nM PMA (Sigma‐Aldrich) for 24 h to induce their differentiation into macrophage‐like cells, followed by washing three times with PBS. Transient transfections were performed using calcium phosphate (Clontech) in 293T, according to the manufacturer’s instructions. RAW264.7 and THP‐1 stable cell lines were generated by transfections were performed using Lipofectamine 3000 (Invitrogen) and then a standard selection protocol with 400–800 μg/ml of G418.
10
Gene Transfection Protocols for Cell Lines
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