KG1a cells were electroporated with non-targeting scrambled siRNA (Dharmacon #D-001810–10-20), siRNA targeting ASAH1 (Dharmacon #L-005228–03-0010) or MCL-1S (Dharmacon #CTM-481502) using the Neon Transfection System (Invitrogen) according to the manufacturer’s protocol with the following parameters: 3 × 107 cells per ml, 1700 pulse voltage, and 20 ms pulse width for a single pulse. AC knockdown cells were harvested 48 hours after transfection for analysis by western blot. Mcl-1S knockdown cells were re-plated to 2.5 × 105 cells per ml and treated with SACLAC 24 hours after electroporation. Control- and SACLAC-treated cells were harvested 48 hours after SACLAC treatment, corresponding to 72 hours after electroporation. HL-60/VCR cells were transduced with vectors expressing shRNA to target AC or GFP control as previously described (23 (link)).
D 001810 10 20
Manufactured by Horizon Discovery
Sourced in United States
The D-001810-10-20 is a laboratory equipment product manufactured by Horizon Discovery. It is designed to perform a core function, but a detailed description cannot be provided without the risk of extrapolation or interpretation. Therefore, a concise, unbiased, and factual description is not available.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (9 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
M50 super 8x topflash, by Addgene (2 mentions)
Prl rluc myc, by Promega (2 mentions)
L 020431 00 0005, by Horizon Discovery (2 mentions)
Recombinant wnt3a, by R&D Systems (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
On targetplus, by Horizon Discovery (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Am51331, by Thermo Fisher Scientific (1 mentions)
Dharmafect 1, by Horizon Discovery (1 mentions)
Control sirna, by Horizon Discovery (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Viromer red, by BioNTech (1 mentions)
Kc57 rd1, by Beckman Coulter (1 mentions)
Smartpool sirna, by Horizon Discovery (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
24 protocols using d 001810 10 20
1
Knockdown of ASAH1 and MCL-1S in KG1a Cells
2
siRNA-Mediated Knockdown in Cell Lines
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The following siRNA oligonucleotides were used for RNA interference (RNAi): NLRP3, 114548 (Dharmacon); ASC, 1027416 (Qiagen); AM51331 (Ambion), siRNA 44415 (siASC 2); 4392420 (Ambion), siRNA s26508 (siASC 3); and negative-control siRNA D-001810-10-20 (Dharmacon). siRNA duplexes were reverse transfected into cells using Lipofectamine RNAiMAX (17-0618-0; Invitrogen) on the day of plating (24 h before infection or treatment) according to the manufacturer’s instructions. Two subsequent transfections were performed on adherent cells: the first one before infection and the second one on day 3 postinfection.
3
Wnt/β-Catenin Signaling Modulation
4
siRNA Transfection for EGR1 Knockdown
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Transfection of siRNAs was performed in suspension using Lipofectamine RNAiMAX Transfection Reagent (no. 13778, Invitrogen). Human siRNA EGR1 (Dharmacon, L-006526-00-0005) and nontargeting siRNA pool (Dharmacon, D-001810-10-20) were used at the final concentration of 10 nM. Knock-down efficiency was assessed after 72 hours through immunoblot and qPCR analysis.
5
ARID1A-BAF250a Silencing and HIV Latency
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Pre-designed siRNA pools targeting transcripts of the human ARID1a-BAF250a (L-017,263-00-0005) and non-target control siRNA pool (D-001,810-10-20) were purchased from Dharmacon (Dharmacon, Etten-Leur, The Netherlands). Small-interfering RNAs were delivered by nucleofection using Amaxa Nucleofector (Amaxa AG-Lonza, Cologne, Germany) as previously described (Rafati et al., 2011 ). Briefly, cells were split to 3 × 105 cells/ml one day before nucleofection. Five million cells were centrifuged at 200 g for 10 min at room temperature, re-suspended in 100 μl of solution R, and nucleofected with 2 μM siRNA using program O28. Nucleofected cells were re-suspended in 500 μl of pre-warmed, serum-free antibiotic-free RPMI at 37 °C for 15 min and then plated in 4 ml of pre-warmed complete media. Seventy-two hours post-nucleofection cells were treated with SAHA [350 nM] or Prostratin [100 nM]. LTR-driven GFP expression was assessed after 24 and 48 h after treatment by FACS. RNA and protein for RT-qPCR and Western blot analysis were isolated 96 h after nucleofection.
6
Characterizing Cyclin D1 Function
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A validated pool of 4 siRNA constructs directed against the N-terminus of the murine cyclin D1 transcript or scramble control was purchased from Dharmacon (L-042441-00-0020 and D-001810-10-20) and transfected into Ccnd1+/+ or Ccnd1KI/KI lines using the Dharmacon 1 transfection reagent. Forty-eight hours post-transfection, cells were harvested and utilized for immunoblot, growth, and immunofluorescence assays.
7
siRNA-mediated CDK8 knockdown assay
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CDK8 was targeted with ON-TARGETplus SMARTpool L-003242-00-0005 and the negative control was D-001810-10-20 (both from Dharmacon, Lafayette, USA). The DharmaFECT siRNA protocol was followed. Briefly, cell lines were seeded in a 6-well plate at 500,000 cells/well and incubated with E medium + 5% FBS. Twenty-four hours post seeding, the siRNA was diluted to 35 nM in Opti-MEM (Gibco, Grand Island, USA, #11058-021) using DharmaFECT1 (Dharmacon, Lafayette, USA, T-2001-02) at a concentration of 5 μL/mL and added to cells incubated with E medium + 5% FBS, according to the manufacturer’s protocol. Cells were harvested for RNA or protein as described above 72 h post-transfection.
8
Recombinant LRG1 Protein Expression and Knockdown
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Human LRG1 (NM_052972) carrying a 6xHis tag expression vector, pcDNA3.1-LRG1, was generated as previously described (11 (link)). rhLRG1 protein was expressed in Freestyle 293 T cells (Invitrogen) and purified as previously described (11 (link)). siRNA oligonucleotides (cat. no. L-015179-01; Dharmacon) were used for LRG1 gene knockdown, while control siRNA (D-001810-10-20; Dharmacon) was used as a negative control. Transfection was performed using Lipofectamine 2000 (Invitrogen) for HaCaT cells and RNAiMAX (Invitrogen) for dHL-60 cells according to the manufacturer’s protocol.
9
siRNA Knockdown in Macrophages
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Scrambled siRNA control (Dharmacon, D-001810-10-20), siRNAs against Gramd1b (Dharmacon, L-040247-01-0005) and Brcc3 (Dharmacon, L-060013-01-0010) were transfected into BMDMs using Lipofectamine RNAiMAX (Life Technologies) at 40 nM of siRNA in 24-well or 96 well plates following the manufacturer’s instructions.
10
Knockdown of Autophagy Receptors and Viral Antigen Expression
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HeLa‐CIITA cells were incubated in 6‐well plates using 2–4.105 cells/well using OPTIMEM (Gibco) complemented with 10% FBS and 1% penicillin/streptomycin. Twenty‐four hours later, cells were transfected with 40 pmol of siRNA targeting NDP52 (L‐010637‐00‐0005), OPTN (L‐016269‐00‐0005), p62 (L‐010230‐00‐0005), T6BP (L‐016892‐00‐0020 Dharmacon or SI02781296, Qiagen), CANX (SI02663367 and SI02757300, Qiagen) or a scrambled siRNA as control (D‐001810‐10‐20, Dharmacon), using Lipofectamine RNAiMax (13778–150, Thermo Fisher) as transfection reagent. After 24 h of transfection, cells were transfected with the cDNA encoding the viral antigens (1 μg per well of a 6‐well plate) using Viromer RED (Lipocalyx) and following manufacturer instructions. Twenty‐four hours later, Gag and pp65 expressions were assessed using anti‐Gag antibody (KC57‐RD1, Beckman‐Coulter) and anti‐pp65 antibody (mouse, Argene) combined with goat anti‐mouse antibody (AF488, Thermo Fisher), respectively.
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