For lentivirus production of CRISPR libraries, transgenes or single guide HEK293T cells were transfected with, HEK293T cells (ATCC) were seeded in T175 cell culture flasks in DMEM (Gibco) supplemented with 10% FBS and 1% Penicillin/Streptomycin and grown up to 70% confluency. HEK293T cells were transfected with the following mixture: 10.4 µg psPAX2-Plasmid, 3.5 µg pMD2.G, 13.8 µg lentiviral vector of interest (see section “Plasmids”) in a volume of 1000 µl Opti-MEM (Gibco) in tube 1. In a second tube, 138 µl 1 mg/mL PEI (Polysciences) was mixed with 862 µl Opti-MEM. Both tubes were incubated at room temperature for 5 min, mixed, and incubated again 20 min at room temperature and added to the cells in the evening. The next morning, the medium was refreshed. After 48 and 72 h, the supernatant was harvested, filtered with 0.45 µm syringe filters (Sarstedt) and concentrated by centrifugation at 24,000 × g for 2 h. Plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12559) were gifts from Didier Trono.
Opti mem
Manufactured by Polysciences
Sourced in United States, Germany
Opti-MEM is a serum-reduced cell culture medium formulated to support the growth and maintenance of a variety of mammalian cell lines. It is designed to be used in conjunction with reduced serum levels, which can help minimize the effects of serum components on experimental outcomes.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (31 mentions)
Polyethylenimine (pei), by Polysciences (12 mentions)
Polybrene, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Pei max, by Polysciences (2 mentions)
Puromycin, by InvivoGen (2 mentions)
Polyethyleneimine max, by Polysciences (2 mentions)
Hitrap kappaselect column, by GE Healthcare (2 mentions)
Pei 25k, by Polysciences (2 mentions)
Pmd2 g, by Thermo Fisher Scientific (2 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (2 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Calcitriol, by Merck Group (1 mentions)
Bexarotene, by Cayman Chemical (1 mentions)
Sh800z cell sorter, by Sony (1 mentions)
Nepa21, by Nepa Gene (1 mentions)
30 protocols using opti mem
1
Lentiviral Vector Production in HEK293T
2
Inducible Reporter Gene in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells were plated into 6-well or 12-well plates at a concentration of 1.3 × 105/mL. On the following day, the cells were incubated with a mixture of 1 μg of DNA of the hyPBase, and the transposon donor vector (at a ratio of 1:3) and 4 μg of polyethylenimine (Polysciences, Warrington, PA, USA) in 100 μL of Opti-MEM for 1 mL of the culture volume. After cell propagation, the EGFP-positive population, which was considered to be expressed constitutively, was removed by three rounds of cell sorting on an SH800Z cell sorter (Sony, Tokyo, Japan). Next, the cells propagated on a 10-cm dish scale were stimulated with 10 μM bexarotene (No. 11571, Cayman Chemical, Ann Arbor, MI, USA) or 150 nM calcitriol (D1530, Sigma, Tokyo, Japan) overnight at 37 °C in a CO2 incubator; subsequently, those that appeared in the EGFP-high fraction were single-cell sorted into 96-well plates. After propagation of those cells, their reagent responsiveness was checked under a fluorescence microscope. Whenever multiple clones were obtained from the same parental cells, the genomes were subjected to a splinkerette PCR analysis, as described in [1 (link)], to select independent clones as assumed from their band patterns.
3
Efficient Transfection of Mammary Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 1 × 106 cells of the sorted MaSC-enriched fraction from about eight mice were suspended and washed in Opti-MEM (Life Technologies, Carlsbad, CA, USA) twice. Then, 10 μg of donor and helper vector DNA were mixed at a ratio of 3:1 with the MECs in Opti-MEM, and subjected to electroporation using NEPA21 (NEPAGENE, Chiba, Tokyo) with a 2-mm gap electrode cuvette (NEPAGENE, EC-002S). The settings for this electroporation are shown in Additional file 1 (Table S1). After electroporation, the cell suspension was immediately suspended in culture medium.
For transfection into NMuMG, dissociated cells were plated on six-well plates at 20–30% confluence a day before transfection. Donor and helper vectors were introduced at an OD260 ratio of 3:1 by pouring the mixture of 4 μg of DNA and 8–12 μg of polyethylenimine (Polysciences, Warrington, PA) into 200 μL of Opti-MEM.
For transfection into NMuMG, dissociated cells were plated on six-well plates at 20–30% confluence a day before transfection. Donor and helper vectors were introduced at an OD260 ratio of 3:1 by pouring the mixture of 4 μg of DNA and 8–12 μg of polyethylenimine (Polysciences, Warrington, PA) into 200 μL of Opti-MEM.
4
TGF-β Signaling Pathway Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells were treated with trypsin, suspended in fresh medium without antibiotics, and seeded onto 6-well culture plates at a cell number of 2 × 105 cells/well. The day after seedings, the pGL4.48[luc2P/SBE/Hygro] vector (2.2 mg; Promega, WI, USA) was diluted with 100 mL Opti-MEM (Life Technologies) and then mixed with 100 μL Opti-MEM containing 6 mL PEI 250,000 (Polysciences, PA, USA) at a concentration of 0.3 g/L. After incubation for 15 min at room temperature, transfection mixtures were added to each well. After 24 h, the cells were detached with trypsin again and re-seeded onto 96-well culture plate at a cell number of 1 × 104 cells/well. After further 1 day incubation, the cells were treated with premixtures of TGF-β1 (PeproTech) and each aptamer or anti-pan-TGF-β antibody (MAB1835, clone no. 1D11, R&D Systems) at indicated concentrations for 3 h, which were diluted with DMEM without serum. The incubated cells were lysed with passive lysis buffer (PLB) (Promega), and the expression levels of luciferases were examined by Dual Luciferase Reporter System (Promega). The luminescent signals were measured with CentroXS3 (link) LB960 (Berthold Technologies, Bad Wildbad, Germany).
5
Lentivirus Production for T Cell Transduction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate lentivirus, 2.5 million low passage HEK293T cells were cultured in DMEM medium and seeded into a 15 cm tissue-culture treated dish. Three days later, 2nd generation LTR-containing donor plasmid, packaging plasmid pCMV-delta8.9 and the envelope plasmid VSV-G were mixed at a ratio of 4:2:1 ratio in unsupplemented Opti-MEM (ThermoFisher) and sterile filtered. This solution was then mixed with polyethyleneimine 25 kDa (Polysciences Inc.), also diluted in Opti-MEM at a DNA:PEI ratio of 1:3. 28 μg of DNA was transfected per 15 cm dish. After two days, supernatants were collected from cells (exchange medium on plates) and filtered through a 0.45 μm PES filter. Supernatants were stored for 1 day at 4 °C until the second batch of supernatant was collected 24 h later. The supernatant containing lentiviral particles was concentrated by ultra-centrifugation at 40’000 x g for 2 h at 4 °C, resuspended in 0.1% BSA in PBS, and frozen to −80 °C. To ease production of antigen specific T cells, virus production for the NY-ESO-1 TCR vector was later outsourced to Vectorbuilder Inc (USA), who provided stocks of > 1*10^9 TU/ml lentivirus produced by PEG precipitation (measured by p24 ELISA).
6
Establishing Stable Cell Lines Expressing or Depleting PD-L1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NIH3T3 cells stably expressing fPD-L1, NIH3T3/fPDL1 were established using retrovirus
transduction. Briefly, PLAT-E cells (7.5 × 105 cells) were seeded in a 6 well
plate, one day before transfection. A mixture of 1.25 µg of pMx-IP-fPDL1-FL#9 was
incubated with 5 µL of 1 µg/mL PEI Max (Polysciences, Warrington, PA, USA) in 62.5 µL of
OPTI-MEM (Thermo Scientific, Yokohama, Japan) for 15 min at room temperature, then added
to the cell culture. Twenty four hours after transfection, medium was replaced with a new
D10 medium. After further 24 hr incubation, the supernatant was collected from transfected
culture and used for viral transduction into NIH3T3 cells as described by [30 (link)]. The transduced cells were cultured for selection
in the presence of 10 µg/mL of puromycin (Sigma-Aldrich Japan K.K., Tokyo, Japan).
FYMp cells stably knocked out of fPD-L1, FYMp-kofPDL1, were established using lentivirus
transduction. Briefly, HEK293T cells (7.5 × 105 cells) were seeded in a 6 well
plate, one day before transfection. A mixture of 0.375 µg of lentiCRISPR-fPDL1#7 was
incubated with 0.5 µg of p8.9QV, 0.375 µg of pCVSVG, and 5 µL of 1 µg/mL PEI Max
(Polysciences, Warrington, PA, USA) in 62.5 µL of OPTI-MEM for 15 min at room temperature,
then added to the cell media. Subsequent steps are the same as above, but 2 µg/mL of
puromycin (Sigma-Aldrich Japan K.K.) was used for selection.
transduction. Briefly, PLAT-E cells (7.5 × 105 cells) were seeded in a 6 well
plate, one day before transfection. A mixture of 1.25 µg of pMx-IP-fPDL1-FL#9 was
incubated with 5 µL of 1 µg/mL PEI Max (Polysciences, Warrington, PA, USA) in 62.5 µL of
OPTI-MEM (Thermo Scientific, Yokohama, Japan) for 15 min at room temperature, then added
to the cell culture. Twenty four hours after transfection, medium was replaced with a new
D10 medium. After further 24 hr incubation, the supernatant was collected from transfected
culture and used for viral transduction into NIH3T3 cells as described by [30 (link)]. The transduced cells were cultured for selection
in the presence of 10 µg/mL of puromycin (Sigma-Aldrich Japan K.K., Tokyo, Japan).
FYMp cells stably knocked out of fPD-L1, FYMp-kofPDL1, were established using lentivirus
transduction. Briefly, HEK293T cells (7.5 × 105 cells) were seeded in a 6 well
plate, one day before transfection. A mixture of 0.375 µg of lentiCRISPR-fPDL1#7 was
incubated with 0.5 µg of p8.9QV, 0.375 µg of pCVSVG, and 5 µL of 1 µg/mL PEI Max
(Polysciences, Warrington, PA, USA) in 62.5 µL of OPTI-MEM for 15 min at room temperature,
then added to the cell media. Subsequent steps are the same as above, but 2 µg/mL of
puromycin (Sigma-Aldrich Japan K.K.) was used for selection.
7
Producing Clade B HIV-1 Pseudoviruses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Clade B (HXB2) HIV-1 PV was used to test the ability of the compounds to inhibit HIV-1 entry into target cells. HIV PVs were produced by transfecting HEK293T-17 cells, using the protocol described by Longo and colleagues [96 (link)]. Briefly, plasmid DNA mix (pCAGGSHXB2, p8.91, and pCSFLW) and transfection reagent polyethyleneimine (PEI) mix from Polysciences, Inc. (Polysciences Inc., Warrington, PA, USA), were prepared in separate Eppendorf tubes containing serum-free media (OptiMEM). The contents of the two tubes were mixed and incubated for about 20 min and afterward added to HEK293T-17 cells. The transfection plate was incubated overnight at 37 °C (5% CO2). The media were changed after 14 h, with culture supernatant containing HIV-1 PVs harvested at 48- and 72-h post-transfection.
8
Lentiviral Knockdown of PLXNC1 in PLC/PRF/5 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lentiviral particles were produced as follows: first, lentiviral PLXNC1 shRNA (TRCN0000060645, Sigma-Aldrich, St. Louis, MO, USA) or control pLKO.1 (Addgene #8453) plasmids were mixed with packaging plasmids pCMV-dR8.2 dvrp (Addgene #8455) and pCMV-VSV-G (Addgene #8454) at a ratio of 1,5:1,5:1 in 250 μl Optimem (Thermo Fisher Scientific, Rockford, IL, USA). Then, a second mixture consisting of the transfection agent PEI (Polysciences, Germany), which was added to 250 μl Optimem at a ratio of 1:3 (DNA μg: PEI μl), was prepared. The two mixtures were assembled in a single tube to generate a transfection reagent, which was used to transfect HEK293T cells after incubation for 20 min at room temperature. After 36 hours, viral particles were harvested from the supernatant of the transfected cells, filtered through 0.45 μm, and stored at -80°C. 1.5x105 PLC/PRF/5 cells plated into a 6-well plate were transduced with viral particles in the presence of 8 μg/ml Polybrene (Thermo Fisher Scientific). The next day, selection of transduced cells was initiated with the addition of 2 μg/ml puromycin (InvivoGen, San Diego, CA, USA) into the culture medium.
9
Recombinant Antibody Production Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PGZL1 HCs and LCs were cloned using Gibson Assembly Enzyme mix (NEB) into expression vectors with the appropriate IgG1, Igκ, or Igλ constant domains49 (link). Antibodies were expressed in FreeStyle 293F cells (Life Technologies Cat#R79007). Briefly, ~ 750 µg DNA (500 µg HC and 250 µg LC plasmid) were added to 25 ml Opti-MEM (Life Technologies, 31985-070), which was mixed with Opti-MEM containing 2250 µg polyethylene imine MAX (molecular weight 40,000 kDa; Polyscience, 24765-1). After incubation for 20 min at room temperature (RT), the transfection mix was added to 1 L cells at a density of ~ 1.2 × 106 cells/ml in FreeStyle293 Expression Medium (Life Technologies, 12338018). The cells were incubated at 37 °C and 8% CO2 for 6 days. After collecting the cells, the supernatant, containing IgG or Fab, was filtered and loaded into a protein A beads column (Thermo Scientific) or HiTrap KappaSelect column (GE Healthcare Life Sciences, 17545812). The column was washed with phosphate-buffered saline (PBS) and eluted with 0.2 M citric acid pH 3.0 or 0.1 M glycine pH 2.7. The fractions were concentrated and the buffer was changed to 20 mM sodium acetate pH 5.5. The Fab was loaded into a Mono S column and was eluted with a 0–60% linear gradient of 1 M sodium chloride and20 mM sodium acetate pH 5.5 buffer. The Fabs were concentrated and stored in 20 mM sodium acetate pH 5.5 at 4 °C.
10
Notch-1 and PTEN Regulate Trastuzumab Response
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!