HepAD38 cells were maintained in Roswell Park Memorial Institute medium (RPMI)-1640 with 20% fetal bovine serum and Huh7 and HepG2-NTCP cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The HepG2-NTCP cells were provided by Dr. Wenhui Li (National Institute of Biological Sciences). The media was supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.1 mM nonessential amino acid under standard culture conditions (5% CO2, 37 °C). PHHs were purchased from Gibco and cultured according to the manufacturer’s protocol. Plasmids were transfected into cells using Mirus TransIT-LT1 reagent (Mirus) according to the manufacturer’s protocol. Lipofectamine RNAiMAX reagent was used for siRNA transfection (Thermo Fisher Scientific) according to the manufacturer’s protocol.
Mirus transit lt1 reagent
Manufactured by Mirus Bio
Sourced in United States
The Mirus TransIT-LT1 reagent is a transfection reagent designed for the delivery of nucleic acids, such as plasmid DNA, siRNA, and mRNA, into a variety of mammalian cell types. The reagent is formulated to facilitate efficient and gentle transfection with minimal cytotoxicity.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem reduced serum media, by Thermo Fisher Scientific (2 mentions)
Pmd2 g, by Addgene (2 mentions)
Prl 3 cdna expression vector, by OriGene (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
100 mm dishes, by Corning (1 mentions)
Polyvinylidene fluoride filter, by Merck Group (1 mentions)
Lentivirus precipitation solution, by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
Sh800s cell sorter, by Sony (1 mentions)
Facs vantage se flow cytometer, by BD (1 mentions)
Recombinant human ifn γ, by R&D Systems (1 mentions)
Recombinant human tnf α, by R&D Systems (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Cycloheximide (chx), by Thermo Fisher Scientific (1 mentions)
Lenti x 293t cells, by Addgene (1 mentions)
Lenti x 293t cells, by Takara Bio (1 mentions)
8 protocols using mirus transit lt1 reagent
1
Cell Culture Protocols for HepAD38, Huh7, and HepG2-NTCP
2
Lentiviral Transduction of HEK293T and A549 Cells
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On day 1, HEK293T cells were seeded into 100mm dishes (Corning). On day 2, cells were ∼50%–70% confluent at the time of transfection. For each dish, 6 μg of pHR vector containing the construct of interest, 4 μg of dR8.91 and 1 μg of pMD2.G (Addgene) were mixed in 1 mL of Opti-MEM reduced serum media (GIBCO) with 30 μL of Mirus TransIT-LT1 reagent and incubated at room temperature for 30 minutes. The transfection complex solution was distributed evenly to HEK293T cultures dropwise. On day 5, lentiviruses are harvested from the supernatant with a sterile syringe and filtered through a 0.22-μm polyvinylidene fluoride filter (Millipore).
For A549 cells, lentivirus precipitation solution (Alstem) was added and precipitated as per the manufacturer’s protocol. One well of a 6-well plate (Corning) of A549 cells at ∼50% confluency was transduced with the precipitated lentivirus. After 2-3 days of growth, the cell supernatant containing the virus was removed and the cells were expanded. Cells were then sorted for mCherry+ cells using a Sony SH800S cell sorter.
For A549 cells, lentivirus precipitation solution (Alstem) was added and precipitated as per the manufacturer’s protocol. One well of a 6-well plate (Corning) of A549 cells at ∼50% confluency was transduced with the precipitated lentivirus. After 2-3 days of growth, the cell supernatant containing the virus was removed and the cells were expanded. Cells were then sorted for mCherry+ cells using a Sony SH800S cell sorter.
3
Stable HEK293 Cell Line Generation
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HEK293
cells were transfected using MIRUS TransIT-LT1 Reagent
(Mirus) and selected for puromycin resistance (2 ug mL–1) after 48 h. For the generation of stable lines, the puromycin resistant
cells were harvested using trypsin (0.05% w/v), resuspended in PBS,
and sorted using a Becton Dickinson FACS Vantage SE Flow Cytometer
(Becton Dickinson) with excitation using the 405 nm laser, and emission
filters were 450/50 nm. The gates were set above an untransfected
control to retain those cells expressing mCer3. The 15 cell lines
were transfected in three batches over three consecutive days and
then sorted in three batches over three days also.
cells were transfected using MIRUS TransIT-LT1 Reagent
(Mirus) and selected for puromycin resistance (2 ug mL–1) after 48 h. For the generation of stable lines, the puromycin resistant
cells were harvested using trypsin (0.05% w/v), resuspended in PBS,
and sorted using a Becton Dickinson FACS Vantage SE Flow Cytometer
(Becton Dickinson) with excitation using the 405 nm laser, and emission
filters were 450/50 nm. The gates were set above an untransfected
control to retain those cells expressing mCer3. The 15 cell lines
were transfected in three batches over three consecutive days and
then sorted in three batches over three days also.
4
PRL-3 Knockdown in Cancer Cells
5
Generation of Reporter Cell Line
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The plasmid encoding Cas9D10A, GFP and gRNAs, the donor plasmid used for recombination, as well as the protocol for validation of the obtained cell line were generated previously (Burov et al., 2021 (link)). SW480 cells were co-transfected with the plasmids using Mirus TransIT-LT1 reagent (Mirus Bio LLC, Madison, WI, USA) according to manufacturer’s instructions. Forty-eight hours post transfection cells were washed with PBS, detached from the plates using trypsin-EDTA solution (Pan-Eko, Moscow, Russia), centrifuged and resuspended in 1 mL of PBS. Then the FACSAria III cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) was used to obtain the population of cells with considerable GFP fluorescence. After that, cells were cultured for 2 weeks and then stimulated with 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF-α (both from R&D systems, Minneapolis, MN, USA) for 72 h. Finally, cells with mCherry fluorescence were collected using the FACSAria III cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) and propagated as described above.
6
Transfection and Chemical Treatment Protocols
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siRNA transfections were performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's protocol. Sequences of siRNAs used in this study are described in Table S1 . Plasmid transfections were performed using Mirus TransIT-LT1 reagent (Mirus Bio) according to the manufacturer's protocol. CHX (Thermo Fisher Scientific) was used at a concentration of 100 μg/ml for the indicated times. APH, HU, and thymidine concentration and treatment duration are described in the legends to the figures.
7
SARS-CoV-2 Lentiviral Knockdown Assay
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On day 0, Lenti-X 293T cells (Takara Bio) were seeded to 1:6 from a 100 mm dish into a 150 mm dish. The following day, Lenti-X 293T cells were transfected with 27.18 μg of SARS-CoV-2 reporter, 23.76 μg of dR8.91 and 2.97 μg of pMD2.G (Addgene) in 4.5 mL of Opti-MEM reduced serum media (GIBCO) with 150 μL of Mirus TransIT-LT1 reagent. 30,000 Cas13d A549 or wild-type A549 cells were also seeded in 24-well plates. On day 2, A549 cells were transfected with pools of targeting crRNAs or a non-targeting guide (50 μL Opti-MEM reduced serum media, 0.5 μg crRNA pool, 1 μL Mirus LT1 per well). On day 3, lentiviruses were harvested from the supernatant with a sterile syringe and filtered through a 0.22-μm polyvinylidene fluoride filter and precipitated with lentivirus precipitation solution and resuspended in 7 mL DMEM media. The media on the cells was replaced with either fresh media for the compensation controls or 0.5 mL virus-containing media at the following dilutions for the experimental conditions: undiluted, 1/2 dilution, 1/3 dilution, and 1/4 dilution. On day 4, the media on all the cells was replaced with 0.5 mL fresh DMEM. On day 5, cells were measured by flow cytometry and assessed for GFP knockdown. Statistical analyses were done using a two-sided t test with unequal variance in Excel to calculate p values.
8
Genetic Manipulation of PRL-3 and Signaling Pathways
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PRL-3 cDNA expression vector was purchased from Origene (Rockville, MD, USA; Cat. #SC308739). Transfections were carried out using Mirus TransIT LT1 reagent according to the manufacturer's instructions (Mirus Bio, Madison, WI, USA). Individual pLKO.1 lentiviral shRNA clones were obtained from the University of Colorado Cancer Center Functional Genomics Shared Resource. The RNAi Consortium identifiers are: TRCN0000010661 (shPRL-3 #1), TRCN0000355597 (shPRL-3 #2), TRCN0000014683 (shp65 #1), TRCN0000014684 (shp65 #2), TRCN0000359596 (shTNF-R1 #1) and TRCN0000359597 (shTNF-R1 #2). Transduced cells were selected in medium containing 2.5 ug/ml puromycin. PRL-3 shRNAs were previously validated.6 (link)
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