C57Bl/6 bone marrow derived BMDC were harvested on day 8. For efficient RNA interference, cells were transfected with mouse GR siRNA (Dharmacon) or non-silencing control scrambled (mock) siRNA (RL, Dharmacon) by using the Amaxa® Mouse Dendritic Cell Nucleofector® Kit (Lonza). Sixteen hours after RNA interference, cells were pre-treated with DEX or CpdA or solvent control and stimulated with LPS or PBS as a control.
Amaxa mouse dendritic cell nucleofector kit
Manufactured by Lonza
Sourced in Switzerland
The Amaxa Mouse Dendritic Cell Nucleofector Kit is a laboratory equipment product designed for the transfection of mouse dendritic cells. It provides a solution for introducing nucleic acids into these specific cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase kit, by Promega (1 mentions)
Magnetic bead separation, by Miltenyi Biotec (1 mentions)
Negative selection magnetic bead isolation kit, by Miltenyi Biotec (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Isotype matched control, by R&D Systems (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Spi1 sirna, by Thermo Fisher Scientific (1 mentions)
Irf8 sirna, by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Nucleofector 2b, by Lonza (1 mentions)
7 protocols using amaxa mouse dendritic cell nucleofector kit
1
GR Knockdown in Murine BMDCs
2
Dendritic Cell Transfection for Migration
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DCs were transfected with SMARTpool: ON-TARGETplus siRNAs specific for Arpc4, LMNA or Sun1 (Thermo scientific) using the Amaxa Mouse Dendritic Cell Nucleofector Kit (Lonza). Briefly, 3 × 106 DCs collected at day 7 of differentiation were transfected in 100 μl of Amaxa solution containing 1 μM of the desired siRNA. Transfected cells were cultured during 48–72 h in DC medium and then used to perform the desired experiment.
We noted that systematically, and for unknown reasons, control siRNA-electroporated DCs migrate faster than non-electroporated ones. Thus, they have a higher rate of passage through constrictions and a lower passage time (Supplementary Fig. 2 compared with Supplementary Fig. 12 ). For this reason, values should be compared with their internal control (either non-electroporated or electroporated).
We noted that systematically, and for unknown reasons, control siRNA-electroporated DCs migrate faster than non-electroporated ones. Thus, they have a higher rate of passage through constrictions and a lower passage time (
3
Exosome-mediated miRNA Regulation in BMDCs
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Knockout BMDCs (2.5 × 105) were transfected with 3′-UTR luciferase reporter constructs (for mir-155−/−: pmiReport, Bach1, Bach1 155 mutant, 2mer29 (link); (for miR-146−/−: pmiReport, Traf6, Traf6 146a mutant45 (link)) using Lonza's Amaxa Mouse Dendritic Cell Nucleofector Kit, according to manufacturer's instructions. After 6 h of nucleofection, BMDCs were treated with or without Wt BMDC-derived exosomes and luciferase activity was measured 24 h later using a Dual Luciferase Kit (Promega). Luciferase repression of exosome-treated BMDCs compared with no exosome treatment was calculated and graphed as per cent change in luciferase activity. Renilla luciferase was used to normalize firefly luciferase values. 2 μg of each construct was transfected into BMDCs.
4
Generating Bone Marrow-Derived Dendritic Cells
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Bone marrow-derived DC (BMDC) were generated, LPS-matured and OVA-pulsed as previously described
24 (link). Briefly, bone-marrow (BM) cells were extracted from the femur and tibia of mice. To generate BMDC, BM cells were cultured over 7 days in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Gibco) in the presence of 20 ng/ml GM-CSF (BioSource). For all experiments, BM DCs were CD11c selected using magnetic bead separation (Miltenyi Biotech). To induce DC activation, CD11c+ DCs were matured overnight with 100ng/ml LPS (Sigma). BMDC were blocked for 30 min at 37°C with anti-IL-15Rα (AF551) or isotype-matched controls (both R&D Systems, 20μg/ml unless otherwise stated). DC were nucleoporated with 5μg lentiviral plasmid DNA using the Amaxa Mouse Dendritic Cell Nucleofector Kit (Lonza). Unsorted cells were used for experiments. Splenic CD4+ T cells were isolated using a negative selection magnetic bead isolation kit (Miltenyi Biotech). For proliferation experiments, CD4+ T cells were labeled with 5μM CFSE dye for 20 min at 37°C then washed before co-culture.
24 (link). Briefly, bone-marrow (BM) cells were extracted from the femur and tibia of mice. To generate BMDC, BM cells were cultured over 7 days in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Gibco) in the presence of 20 ng/ml GM-CSF (BioSource). For all experiments, BM DCs were CD11c selected using magnetic bead separation (Miltenyi Biotech). To induce DC activation, CD11c+ DCs were matured overnight with 100ng/ml LPS (Sigma). BMDC were blocked for 30 min at 37°C with anti-IL-15Rα (AF551) or isotype-matched controls (both R&D Systems, 20μg/ml unless otherwise stated). DC were nucleoporated with 5μg lentiviral plasmid DNA using the Amaxa Mouse Dendritic Cell Nucleofector Kit (Lonza). Unsorted cells were used for experiments. Splenic CD4+ T cells were isolated using a negative selection magnetic bead isolation kit (Miltenyi Biotech). For proliferation experiments, CD4+ T cells were labeled with 5μM CFSE dye for 20 min at 37°C then washed before co-culture.
5
Electroporation of Dendritic Cells with SIVmac239 Env
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DC were electroporated using the Amaxa Mouse Dendritic Cell Nucleofector Kit (Lonza). Briefly, 2X106 DC were resuspended in 100 μl of Nucleofector solution in the presence of 16 μg of plasmid DNA encoding SIVmac239 gp160 Env (plasmid 99S).34,35 (link) The mixture was transferred to an Amaxa certified cuvette and electroporated with an Amaxa Nucleofector apparatus according to the manufacturer instructions. Following electroporation, 400 μl culture medium were added to the mixture and the cells were immediately transferred to a 24-well culture plate containing 500 μl prewarmed culture medium and incubated at 37°C in 5% CO2.
6
Transfection of Bone-Marrow Derived Dendritic Cells
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Bone-marrow derived dendritic cells (BMDCs) were transfected using the Amaxa mouse Dendritic Cell Nucleofector Kit (Lonza), according to the manufacturer's protocol. BMDCs collected on day 6 of differentiation (3 × 106) were transfected in 100 μl of Amaxa solution containing 2 μg of plasmid (empty vector: pCDNA3; Ii full length or IiL7AL17A mutant, kindly provided by Oddmund Bakke). Transfected cells were used for 6–16 h after transfection. Transfection rate was ∼40%.
7
Silencing Macrophage Transcription Factors
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SPI1 siRNA (HSS186060), IRF8 siRNA (HSS105171), Spi1 siRNA (MSS247676), Irf8 siRNA (MSS236848), and a Stealth RNAi siRNA negative control set were purchased from Thermo Fisher Scientific (Waltham, MA, USA). THP-1 cells (2 × 106 cells) suspended in R buffer were mixed with 200 pmol siRNA and then transfected with the Neon transfection system (Thermo Fisher Scientific) setting program No.5. Transfection into BMDMs and human macrophages was performed with Nucleofector 2b (Lonza, Basel, Switzerland) using the Amaxa Mouse Dendritic Cell Nucleofector Kit (Lonza), as previously described (18 (link)).
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