Dharmacon siRNA reagent (#G-005805-01) was delivered into H23 cell through Reverse transfection method67 (link). Briefly, siRNA and lipid (Lipofectamine® 2000 Reagent, 11668019) were mixed and added to 96-well plates. Then, H23 cells were added to the plates (10,000 cells per well). Cell proliferation rate was measured on day 3 by CCK8 assay (CK04-20, Dojindo Molecular Technologies, Inc.). During screening, siRNAs targeting EGFP (5′- CGTGATCTTCACCGACAAGAT-3′) are included in the upper-most and lower-most rows of each plate, which served as negative control. The screening was conducted once.
Sirna reagent
Manufactured by Horizon Discovery
Sourced in United States
The SiRNA reagent is a laboratory product designed for RNA interference (RNAi) experiments. It functions as a tool to temporarily suppress the expression of specific genes in target cells, enabling researchers to study the effects of gene knockdown.
Automatically generated - may contain errors
Lab products found in correlation
Ck04 20, by Dojindo Laboratories (1 mentions)
Profection mammalian transfection kit, by Promega (1 mentions)
Victor2 plate reader, by PerkinElmer (1 mentions)
Nt sirna pool, by Horizon Discovery (1 mentions)
Atlantis c3 reverse phase column, by Agilent Technologies (1 mentions)
Agilent 6100 lc ms spectrometer, by Agilent Technologies (1 mentions)
Zorbax 300sb c3 poroshell column, by Agilent Technologies (1 mentions)
Liberty blue microwave peptide synthesizer, by CEM Corporation (1 mentions)
Phenylephrine, by Merck Group (1 mentions)
4 protocols using sirna reagent
1
siRNA-mediated proliferation assay in H23 cells
2
siRNA Screening for Cell Proliferation
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Dharmacon siRNA reagent (#G-005805-01) was used. Reverse transfection method was used to deliver the siRNA to H23 cell67 (link). Briefly, siRNA and lipid (Lipofectamine® 2000 Reagent) were mixed and added to 96-well plates. Then, H23 cells were added to the plates (10,000 cells per well). Cell proliferation rate was measured on day 3 by CCK8 assay. During screening, siRNAs targeting EGFP (5′-CGTGATCTTCACCGACAAGAT-3′) are included in the upper-most and lower-most rows of each plate, which served as negative control. The screening was conducted for one replica.
3
Transfection and Reporter Assays in Primary HESCs
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Primary HESCs at 80% confluency were transfected with DNA vectors or small interfering RNA (siRNA) oligonucleotides by the calcium phosphate co-precipitation method using the ProFection mammalian transfection kit (Promega, Madison, WI) according to the manufacturer's instructions. Reporter assays were done in 96-well plates. The X-box binding protein 1 (pcDNA3/XBP1-luc) reporter construct was a kind gift from Dr. Etsu Tashiro (Keio University, Tokyo, Japan). A constitutively active renilla expression vector (pRL-sv40) served as an internal transfection control. The plates were washed twice in phosphate-buffered saline (PBS) and firefly and Renilla activities were measured using the Luclite luciferase reporter assay system (Luclite, PerkinElmer, Boston, MA) and the luminescence was measured on a Victor II plate reader (PerkinElmer). For gene-silencing studies, HESCs were cultured in 6-well plates until 80% confluency and transiently transfected with 100 nM of the following siRNA reagents (Dharmacon, Lafayette, CO): siCONTROL non-targeting (NT) siRNA pool and HSPA8 siGENOME SMARTpool. All experiments were performed on three or more primary cultures from different endometrial biopsies.
4
Peptide Synthesis and Purification of CXCR4 Transmembrane Domains
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Phenylephrine was purchased from Sigma-Aldrich (St. Louis, MO, USA) and CXCL12 from Protein Foundry, Milwaukee, WI, USA. siRNA reagents were purchased from GE Dharmacon (Lafayette, CO, USA). The peptide analogs of transmembrane helix 2 (TM2) (LLFVITLPFWAVDAVANWYFGNDD-NH2) and TM4 (VYVGVWIPALLLTIPDFIFANDD-OH) of CXCR4 were generated using the Fmoc protected amino acids in a solid-phase synthesis on a 433A Applied Biosciences Peptide Synthesizer and Liberty Blue Microwave peptide synthesizer (CEM Corporation, Charlotte, NC, USA) using Fmoc chemistry. The peptides were cleaved from the resin and deprotected with a mixture of 90.0% (v/v) trifluoroacetic acid (TFA) with 2.5% water, 2.5% triisopropyl-silane, 2.5% 2,2′-(ethylenedioxy)diethanethiol and 5% thioanisol. Peptides were purified on a preparative (25 mm × 250 mm) Atlantis C3 reverse phase column (Agilent Technologies, Santa Clara, CA, USA) in a 90 min gradient of 0.1% (v/v) trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile, with a 10 mL/min flow rate. The fractions containing peptides were analyzed on Agilent 6100 LC/MS spectrometer (Agilent Technologies, Santa Clara, CA, USA) with the use of a Zorbax 300SB-C3 PoroShell column and a gradient of 5% acetic acid in water and acetonitrile. Fractions that were more than 95% pure were combined and freeze dried.
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