Silencer® Select_FPr1/Silencer® Select_FPr2 siRNA

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	Mouse Neuroblastoma cells (N2a (ECACC 89121404) were obtained from European Collection of Authenticated Cell Cultures (UK). Eagle’s Minimum Essential Medium high glucose (EMEM), Dulbecco’s Modified Eagle’s Medium high glucose (DMEM), all SilencerTM select siRNA duplexes for mouse Fpr1 (siRNA ID s66215), mouse Fpr2 (siRNA ID 66212) plus negative control no.1 siRNA and Lipofectamine RNAiMAX were purchased from ThermoFisher Scientific (UK). All other chemicals used were of reagent grade. N2a cells were cultured in DMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete DMEM). IMR-32 and SH-SY5Y cells were cultured in EMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete EMEM). For siRNA transfections N2a cells were cultured in serum-free antibiotic-free DMEM. Cells were incubated under standard conditions of: 37°C, 5% CO2 in a humidified atmosphere. Cultures were passaged at regular intervals, once at 70–80% confluence.Two siRN A duplexes targeting Fpr1, Fpr2 plus a third negative control knockdown duplex were used. Transfections were carried out using Lipofectamine RNAiMAX in antibiotic-free serum-free DMEM. Lipofectamine RNAiMAX and siRNA duplexes (10μM stock in nuclease-free water) were diluted separately in antibiotic-free serum-free DMEM in a v/v ratio of 1.5:25 and 1:50 respectively. Diluted siRNA was then added to diluted Lipofectamine in a 1:1 ratio and incubated at room temperature for 5min. siRNA-lipid complexes then added to sub-confluent N2a cells; 10μL per well for 96-well plate assays giving a final siRNA concentration of 1pM and 0.3μL Lipofectamine per well, and 50μL per well was used for 24-well plate assays giving a final siRNA concentration of 5pM and 1.5μL Lipofectamine per well. For simultaneous Fpr1 and Fpr2 knockdown, the Fpr1 and Fpr2 siRNA duplexes were added to 0.6μL Lipofectamine (96-well) at a final well concentration of 1pM, and 5pM with 3μL Lipofectamine (24-Well). Transfected cells were then incubated at 37°C, 5% CO2 for 48h before treatment with FPRa14 agonist [28].

Protocol tips

Mouse Neuroblastoma cells (N2a (ECACC 89121404) were obtained from European Collection of Authenticated Cell Cultures (UK). Eagle’s Minimum Essential Medium high glucose (EMEM), Dulbecco’s Modified Eagle’s Medium high glucose (DMEM), all SilencerTM select siRNA duplexes for mouse Fpr1 (siRNA ID s66215), mouse Fpr2 (siRNA ID 66212) plus negative control no.1 siRNA and Lipofectamine RNAiMAX were purchased from ThermoFisher Scientific (UK). All other chemicals used were of reagent grade. N2a cells were cultured in DMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete DMEM). IMR-32 and SH-SY5Y cells were cultured in EMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete EMEM). For siRNA transfections N2a cells were cultured in serum-free antibiotic-free DMEM. Cells were incubated under standard conditions of: 37°C, 5% CO2 in a humidified atmosphere. Cultures were passaged at regular intervals, once at 70–80% confluence.Two siRN A duplexes targeting Fpr1, Fpr2 plus a third negative control knockdown duplex were used. Transfections were carried out using Lipofectamine RNAiMAX in antibiotic-free serum-free DMEM. Lipofectamine RNAiMAX and siRNA duplexes (10μM stock in nuclease-free water) were diluted separately in antibiotic-free serum-free DMEM in a v/v ratio of 1.5:25 and 1:50 respectively. Diluted siRNA was then added to diluted Lipofectamine in a 1:1 ratio and incubated at room temperature for 5min. siRNA-lipid complexes then added to sub-confluent N2a cells; 10μL per well for 96-well plate assays giving a final siRNA concentration of 1pM and 0.3μL Lipofectamine per well, and 50μL per well was used for 24-well plate assays giving a final siRNA concentration of 5pM and 1.5μL Lipofectamine per well. For simultaneous Fpr1 and Fpr2 knockdown, the Fpr1 and Fpr2 siRNA duplexes were added to 0.6μL Lipofectamine (96-well) at a final well concentration of 1pM, and 5pM with 3μL Lipofectamine (24-Well). Transfected cells were then incubated at 37°C, 5% CO2 for 48h before treatment with FPRa14 agonist [28].

Publication protocol

Mouse Neuroblastoma cells (N2a (ECACC 89121404) were obtained from European Collection of Authenticated Cell Cultures (UK). Eagle’s Minimum Essential Medium high glucose (EMEM), Dulbecco’s Modified Eagle’s Medium high glucose (DMEM), all SilencerTM select siRNA duplexes for mouse Fpr1 (siRNA ID s66215), mouse Fpr2 (siRNA ID 66212) plus negative control no.1 siRNA and Lipofectamine RNAiMAX were purchased from ThermoFisher Scientific (UK). All other chemicals used were of reagent grade. N2a cells were cultured in DMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete DMEM). IMR-32 and SH-SY5Y cells were cultured in EMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete EMEM). For siRNA transfections N2a cells were cultured in serum-free antibiotic-free DMEM. Cells were incubated under standard conditions of: 37°C, 5% CO2 in a humidified atmosphere. Cultures were passaged at regular intervals, once at 70–80% confluence.Two siRN A duplexes targeting Fpr1, Fpr2 plus a third negative control knockdown duplex were used. Transfections were carried out using Lipofectamine RNAiMAX in antibiotic-free serum-free DMEM. Lipofectamine RNAiMAX and siRNA duplexes (10μM stock in nuclease-free water) were diluted separately in antibiotic-free serum-free DMEM in a v/v ratio of 1.5:25 and 1:50 respectively. Diluted siRNA was then added to diluted Lipofectamine in a 1:1 ratio and incubated at room temperature for 5min. siRNA-lipid complexes then added to sub-confluent N2a cells; 10μL per well for 96-well plate assays giving a final siRNA concentration of 1pM and 0.3μL Lipofectamine per well, and 50μL per well was used for 24-well plate assays giving a final siRNA concentration of 5pM and 1.5μL Lipofectamine per well. For simultaneous Fpr1 and Fpr2 knockdown, the Fpr1 and Fpr2 siRNA duplexes were added to 0.6μL Lipofectamine (96-well) at a final well concentration of 1pM, and 5pM with 3μL Lipofectamine (24-Well). Transfected cells were then incubated at 37°C, 5% CO2 for 48h before treatment with FPRa14 agonist [28].

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