Twenty micrograms of the CBS variant library (in pDEST_HC_Rec_Bxb_V2), along with an equal mass of Bxb1 recombinase (pCAG-NLS-HA-Bxb1) was transfected into three 15-cm dishes of HEK293T LLP iCasp9 Blast cells (passage 18, 65% confluency) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000008), with volumes scaled based on 3.75 µL reagent per 6 wells. Twenty-four hours later, cells were split 1:2 into 15-cm dishes. Forty-eight hours after transfection, at near full confluency, 2 µg/mL doxycycline and 10 nM AP1903 (MedChemExpress, HY-16046), both solubilized in DMSO, were added for negative selection of non-recombined cells. The next day, dead cells were removed and recombined cells were grown out for an additional 2 days with fresh media containing doxycycline and AP1903. Cells were recovered for 1 day by growth in media without doxycycline and AP1903. Transcription was induced with 2 µg/mL doxycycline for 24 h; cells were stimulated with fresh media for 3 h, and then harvested at 95% confluency.
Ap1903
Manufactured by MedChemExpress
Sourced in United States
AP1903 is a laboratory instrument designed for high-performance liquid chromatography (HPLC) analysis. It provides precise and reliable separation and detection of various chemical compounds. The core function of AP1903 is to facilitate the analytical process by performing chromatographic separation and detection of samples.
Automatically generated - may contain errors
Lab products found in correlation
Fugene hd, by Promega (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Doxycycline dox, by Merck Group (2 mentions)
Doxycycline, by MedChemExpress (2 mentions)
Glucose free dmem, by Thermo Fisher Scientific (2 mentions)
Blasticidin s, by Thermo Fisher Scientific (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Opti mem, by Promega (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Bovine serum, by Merck Group (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Cfx connect detection system, by Bio-Rad (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Mg132, by MedChemExpress (1 mentions)
14 protocols using ap1903
1
Inducible Gene Knockout in HEK293T Cells
2
Maintenance of HEK 293T TetBxb1BFPiCasp9 Clone 12 Cell Line
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The HEK 293 T TetBxb1BFPiCasp9 Clone 12 cell line that was generated previously38 (link) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) (Sigma Aldrich), 0.24 mg/mL streptomycin sulphate (BioChemica), 0.29 mg/mL penicillin G potassium salt (BioChemica), 0.32 mg/mL L-glutamine (Sigma Aldrich) and 2 µg/mL doxycycline (Sigma-Aldrich). Cells were passaged when they reached 70-80% confluency and were detached with 0.25% trypsin (Gibco). The cells tested negative for mycoplasma. Authentication was performed by regular selection for recombinants with 10 nM of AP1903 (MedChemExpress) (see below) and checking for expression of BFP from the Tet-on promoter in non-recombinant cells.
3
Optimizing Inducible Gene Expression
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Cells were transduced with the Ad vector at 3 or 18 pfu per cell and with the AAV vectors at 670 viral genome copies (vg) per cell for 24 h, followed by adding 10 nM AP1903 (MedChem Express, Monmouth Junction, NJ, USA) to cells. Twenty-four hours after treatment, the number of cells and protein expression levels were determined.
4
Stable Transfection of HEK293T Cells
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U2OS cells (ATCC) and HEK293T landing pad cells [38 (link)] were propagated in Dulbecco´s Modified Eagle medium (DMEM), supplemented with 10% bovine serum (Sigma), 5000 IU/mL penicillin, 5 mg/mL streptomycin, 2 mM glutamine and, in the case of HEK293T landing pad cells, 2 μg/mL doxycycline (Dox) (Sigma-Aldrich, D9891), in a humidified atmosphere containing 5% CO2 at 37 °C. Transient transfections were performed with FugeneHD (Promega) in reduced serum medium OptiMEM (Gibco). To generate stable transfectants in the HEK293T landing pad cells, 106 cells in 1 mL DMEM without doxycycline were seeded into 12-well plates. On the next day, the cells were transfected using 0.1 μg of the integrase vector pNLS-Bxb1-recombinase, mixed with 0.4 μg of the pVAMP recombination plasmid, 40 μL OptiMEM and 1.6 μL FugeneHD. Two days after transfection, the cells were treated with 10 nM of AP1903 (MedChemExpress) and 2 μg/mL doxycycline for two days to select for recombinant cells. After another two days of culturing in media with doxycyclin, but without AP1903, the cells were used directly for western blotting, microscopy and flow cytometry.
5
CRISPR-based Genetic Screen in HEK-293T Cells
6
Inducible PRKN Gene Overexpression
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The barcoded PRKN library was recombined into the Tet-on landing pad in the HEK 293T TetBxb1BFPiCasp9 Clone 12 cell line. To this end, 3.5×106 cells were seeded in 10 cm plates and left to grow overnight in media with no doxycycline. After 24 hours, 7.1 μg of the PRKN library plasmid and 0.48 μg of pCAG-NLS-Bxb1 (17.5:1 molar ratio) were diluted with OptiMEM (Thermo Fisher Scientific) to a total volume of 710 μL in an Eppendorf tube. In a second Eppendorf tube, 28.5 μL of Fugene HD (Promega) was added to 685 μL OptiMEM (Thermo Fisher Scientific). The OptiMEM solution containing Fugene HD was added to the DNA/OptiMEM tube. The transfection mix was then added to the seeded cells in a 10 cm dish. About 48 hours, 2 µg/mL doxycycline and 10 nM of AP1903 (MedChemExpress) was added to the cells.
7
Quantitative Analysis of Aire-Induced Gene Expression
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293 T, 4D6 cells or 4D6 stable cells + 0.2–1 μg/ml doxycycline were seeded in a 12-well plate format; cells were harvested 48 h post-transfection or post-induction, respectively. Total RNA was isolated using Direct-zol RNA mini prep kit (Zymo Research) and reverse-transcribed using SuperScript II (Life Technologies) with oligo(dT18). qPCR was performed using Power SYBR Green PCR Master Mix (Invitrogen) on a CFX-Connect detection system (Bio-Rad, Hercules, CA) with Bio-Rad CFX Manager 3.1 software. The expression of Aire-induced genes was normalized against that of the Aire-independent gene GAPDH using the ∆∆Ct method. HPRT1 (Aire-independent gene control) was also normalized against GAPDH. The qPCR primer sequences are listed in Supplementary Table 2 .
For MG132 and AP1903 treatments, after 16 h of transient transfection, the cell media was replaced with DMEM supplemented with 1% FBS, 1% L-glutamine with DMSO, 10 μM MG132 or 5 μM AP1903 (MedChemExpress). For AP1903-treated cells, 3–4 h later, 9% more FBS was supplemented into the media. Cells were harvested 16 h after MG132 or AP1903 treatments.
For MG132 and AP1903 treatments, after 16 h of transient transfection, the cell media was replaced with DMEM supplemented with 1% FBS, 1% L-glutamine with DMSO, 10 μM MG132 or 5 μM AP1903 (MedChemExpress). For AP1903-treated cells, 3–4 h later, 9% more FBS was supplemented into the media. Cells were harvested 16 h after MG132 or AP1903 treatments.
8
Induction of Apoptosis in iPSCs
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iPSCs were cultured in E8 Flex medium for 2 to 4 days in 24‐well, 12‐well, and/or 6‐well plates (Corning) when cell density reached ∼0.15 × 106/cm2 and then exposed to 10 or 50 nM AP1903 (Medchemexpress, Cat. HY‐16046) for the desired amount of time. All cells in each well at the time point were collected and stained with annexin V (AnV) and 7‐amino‐actinomycin D (7‐AAD) (Enzo Life Sciences, Cat. ENZ‐51002‐100) for 15 minutes following the manufacturer's instructions. Cells were then quantified and analyzed by flow cytometry (Attune NxT, Thermo Fisher Scientific) and Flowjo Software. The dead cells were quantified using 100% minus the percent AnV−/7‐AAD− cells in the FSC/SSC gated region after doublet removal. The real percentage of total dead cells in the AP1903 treated condition at some time points could be even higher since majority of the cells became debris and were not included in calculation (Figure 3E,F ).
9
Adenovirus and AAV Transduction Optimization
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Cells were infected with AdV and AAV vectors at 10 infection units (IUs) per cell and 1 × 105 viral genome copies per cells, respectively. Twenty-four hours after infection, 10 nmol/ml AP1903 (MedChem Express, Monmouth Junction, NJ, USA) was added to the cells. Cells were then incubated for 24 hours, and cell numbers, protein expression levels, and gene expression levels were analyzed.
10
PRKN variant integration into HEK293T
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The cDNA of wild-type PRKN or PRKN variants studied in low-throughput were purchased from Genscript. Single PRKN variants were integrated into the Tet-on landing pad in the HEK 293T TetBxb1BFPiCasp9 Clone 12 cell line. First, 3.5 × 106 cells were seeded in 10 cm plates and left to grow overnight in media with no doxycycline. After 24 hours, 3 μg of the PRKN plasmid and 1 μg of pCAG-NLS-Bxb1 (9:1 molar ratio) were added in 400 μL of OptiMEM (Thermo Fisher Scientific). Then, 14 μL of Fugene HD (Promega) was added to the DNA/OptiMEM mixture before adding the entire transfection mix to the seeded cells. About 48 hours after this transfection 2 µg/mL doxycycline and 10 nM of AP1903 (MedChemExpress) was added to the cells, to activate gene expression from the Tet-on promoter and induce apoptosis in cells without recombination at the landing pad locus, respectively.
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