siRNA smart pool targeting EPHA2 (siGENOME Human EPHA2, 5 nmol, M-003116-02-0005), ON-TARGETplus non-targeting control siRNAs (5 nmol, D-001810-01-05), 5x siRNA buffer (100 mL, B-002000-UB-100) and DharmaFECT 1 Transfection Reagent (0.75 mL, T-2001-02) were all purchased from Horizon discovery. Cells at 50–60% confluent were transfected with siRNA using DharmaFECT 1 with the optimum for 40 h and infected with bacteria for 30 h at 37 °C with 5% CO2. ON-TARGETplus non-targeting control siRNAs were used as a control. The knocked-down infected cell lysates were subjected to WB analysis or intracellular bacterial survival assay as described.
B 002000 ub 100
Manufactured by Horizon Discovery
Sourced in United States
The B-002000-UB-100 is a laboratory equipment product. It is a standard piece of equipment used in scientific research and analysis. The core function of this product is to perform specific tasks within a laboratory environment. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (4 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (3 mentions)
B 005000 100, by Horizon Discovery (2 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (2 mentions)
On targetplus non targeting control sirna, by Horizon Discovery (1 mentions)
Sirna buffer, by Horizon Discovery (1 mentions)
Smartpool sirna, by Horizon Discovery (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Thymidine, by Merck Group (1 mentions)
Accell sirna delivery medium, by Horizon Discovery (1 mentions)
Mouse t cell nucleofector kit, by Lonza (1 mentions)
Amaxa nucleofector device, by Lonza (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Accell non targeting control sirna, by Horizon Discovery (1 mentions)
Accell delivery medium, by Horizon Discovery (1 mentions)
On targetplus non targeting pool, by Horizon Discovery (1 mentions)
Vernier stereotaxic instrument, by Leica (1 mentions)
Neurofect transfection reagent, by Genlantis (1 mentions)
14 protocols using b 002000 ub 100
1
EPHA2 Knockdown and Bacterial Infection
2
siRNA-Mediated Knockdown of β-Arrestin 1 in Mouse Islets
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For β-arrestin 1 (Barr1) siRNA studies, CAMPER primary mouse islets were dispersed by trituration in 0.05% trypsin/EDTA for 3 min at 37°C and seeded at the center of poly-d -lysine–coated glass-bottom dishes. Dispersed islets were allowed to recover overnight before treatment with Accell mouse Arrb1 (109689) siRNA-SMARTpool (E-040976-00-0005, Horizon Discovery) or nontargeting control siRNA (D-001910-01-05, Horizon Discovery) according to the manufacturer’s instructions using the recommended siRNA buffer (B-002000-UB-100, Horizon Discovery) and serum-free delivery medium (B-005000-100, Horizon Discovery). Studies took place 72 hours after siRNA treatment addition.
3
Reverse Transfection for RNAi Depletion
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RNAi depletion by reverse-transfection was conducted in MCF10A cells, hTERT RPE-1 cells, and HeLa cells as previously reported (54–56 (link)). Briefly, cells were reverse-transfected into an arrayed 384-well plate library containing small interfering RNA (siRNA) or miRIDIAN microRNA mimic constructs (Horizon Discovery, Supplementary Tables S1 -S7 ) using Opti-MEM (Gibco 31985070) and Lipofectamine RNAiMAX transfection reagent (Invitrogen 13778150). Assay-ready plates containing 10 μl of 100 nM microRNA mimics resuspended in 1X siRNA buffer (Horizon Discovery B-002000-UB-100) were prepared from master library 384-well plates (Horizon Discovery, 0.1 nmol scale) and stored at −80°C. Plates were prepared with control siRNAs (siNT, siNOL11, siKIF11 or siPOLR1A) for reverse-transfection at a final 20 nM siRNA/microRNA mimic concentration as described (56 (link)), at a seeding density of 3000 MCF10A or HeLa cells/well or 4000 hTERT RPE-1 cells/well.
4
CD22 Knockdown Using Accell siRNAs
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Accell siRNAs against human CD22 (set of 4) were synthesized by Dharmacon (Horizon Discovery, EQ-019501-00-0002). 5× siRNA buffer (Horizon Discovery, B-002000-UB-100) combined with sterile RNase-free water was used to prepare 1× siRNA buffer. Lyophilized siRNA was reconstituted to 100 μM siRNA solution in 1× siRNA buffer. According to the manufacturer’s experimental conditions, siRNA medium was prepared by adding 1 μM siRNA solution to 1× Accell delivery medium (Horizon Discovery, B-005000-100). Finally, cells were incubated either with siRNA medium or Accell delivery medium alone at 37°C with 5% CO2 for 96 h.
5
siRNA Transfection and D089 Treatment
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siRNA transfection was performed following manufacturer’s reverse transfection protocols for DharmaFECT 1 transfection reagent (GE Dharmacon, Lafayette, CO, USA) in a 96-well or 6 well plates. The siRNAs used in the study from Dharmacon Inc. are listed below (Table 2 ).
Briefly, siRNAs were resuspended in 1× siRNA Resuspension buffer (GE Dharmacon B-002000-UB-100) and used at a final concentration of 50 nM in OptiMEM. Transfection reagents and siRNA were mixed and incubated at 25 °C for 20 min to form siRNA-liposome complexes. L363 cells (0.5 × 104 cells/well (96-well plate) or 0.5 × 105 cells/well (6-well plate)) were added with the transfection mixture; after for 24 h, complete growth media containing D089 or DMSO (0.1% final) were added to each well and incubated at 37 °C for 48 h.
Briefly, siRNAs were resuspended in 1× siRNA Resuspension buffer (GE Dharmacon B-002000-UB-100) and used at a final concentration of 50 nM in OptiMEM. Transfection reagents and siRNA were mixed and incubated at 25 °C for 20 min to form siRNA-liposome complexes. L363 cells (0.5 × 104 cells/well (96-well plate) or 0.5 × 105 cells/well (6-well plate)) were added with the transfection mixture; after for 24 h, complete growth media containing D089 or DMSO (0.1% final) were added to each well and incubated at 37 °C for 48 h.
6
Downregulation of Cand1 in Prostate Cancer
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For downregulation of Cand1 in PCa cells, a pool of four targeting siRNAs (On-Targetplus Human Cand1siRNA smart pool, L-015562-01, Dharmacon, Vienna, Austria) was used: GACUUUAGGUUUAUGGCUA (J-015562-09), CGUGCAACAUGUACAACUA (J-015562-10), CAACAAGAACCUACAUACA (J-015562-11), CAUAACAAGCCAUCAUUAA (J-015562-12). A pool of 4 non-targeting siRNAs (ON-Targetplus Non-targeting Control Pool, D-001810-10-20, Dharmacon, Vienna, Austria) served as negative control (siCtrl). Both, siCand1 and siCtrl were resuspended in 1× siRNA Buffer (B-002000-UB-100, Dharmacon, Vienna, Austria). Transfection of targeting or control siRNAs (50 nM) was performed using Lipofectamine2000 transfection reagent (Invitrogen, Lofer, Austria) according to the manufacturer´s instruction. Target gene downregulation was confirmed by qRT-PCR and Western blot analysis. When performing functional experiments (p21 expression levels and SKP Elisa), the cell cycle was synchronized with a thymidine block using 2 mM thymidine (Sigma Aldrich, Vienna, Austria) for 18 hours.
7
Nucleofection and siRNA Silencing in OT1 T Cells
8
Eif2ak2 Knockdown in BMDMs
9
APOBEC and DNASE1 siRNA Knockdown
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N/TERT KCs were plated in 96-well plates (30,000 cells/well) and incubated at 37°C with 5% CO2 overnight. Accell siRNA (100 μM; Dharmacon: APOBEC3A, E-017432-00-0005; APOBEC3B, E-017322-01-0005; APOBEC3G, E-013072-00-0005; APOBEC3H, E-019144-00-0005; DNASE1, E-016280-00-0005) was prepared in 1× siRNA buffer (Dharmacon, B-002000-UB-100). One microliter of 100 μM siRNA was diluted with 100 μL Accell delivery medium (Dharmacon, B-005000) for each well of 96-well plates. The growth medium was removed from the cells and 100 μL of the appropriate delivery mix with siRNA was added to each well and the plate was incubated at 37°C with 5% CO2. Accell Non-targeting Control siRNA (Dharmacon, D-001910-01-05) was used as a negative control. After 72 hours, cells were harvested for RNA preparation. RNA isolation and qRT-PCR were as described above.
10
Airway Epithelial Cell Knockdown
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All siRNAs were obtained from Dharmacon/Horizon Discovery: Accell siRNA SMARTPool Human Non-Targeting Control Pool (D-001910-10-50); Accell siRNA SMARTPool Human EZR (E−017370-00-0020); and Accell siRNA SMARTPool Human PAK4 (E−003615-00-0020). siRNAs were resuspended to 100 μM using 1× siRNA buffer (Dharmacon, B-002000-UB-100) and incubated at room temperature for 1 h on a shaker. Passive transfections were performed by adding 1 μM Accell siRNA final concentration to differentiation medium of ALI cultures after 21 days of ALI culture. siRNA concentration was maintained throughout the differentiation protocol with fresh siRNA added with each refeed over the 3 weeks.
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