Human monocyte nucleofector buffer

Manufactured by Lonza

Sourced in Germany

The Human Monocyte Nucleofector buffer is a specialized solution designed for the nucleofection of human monocytes. It is formulated to optimize the delivery of genetic material into these cells, enabling efficient transfection and subsequent analysis or experimentation.

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Lab products found in correlation

Nucleofector kit, by Lonza (2 mentions) On targetplus smartpool, by Horizon Discovery (2 mentions) On targetplus smartpool sirna, by Horizon Discovery (1 mentions) Recombinant human m csf, by Thermo Fisher Scientific (1 mentions) Non targeting sirna, by GenePharma (1 mentions) 4d nucleofector platform, by Lonza (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Corning (1 mentions) Amaxa nucleofector system, by Lonza (1 mentions) Recombinant human m csf, by R&D Systems (1 mentions) Amaxa nucleofector 1, by Lonza (1 mentions)

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Nucleofector buffer (2 citations)

9 protocols using human monocyte nucleofector buffer

Monocyte Nucleofection for Gene Silencing

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Immediately after isolation, primary human monocytes were nucleofected with On-Target Plus SMARTpool siRNA purchased from Dharmacon Inc. specific for IL7R, MAF, or MAP3K3. Nontargeting siRNA from GenePharma was used as a control. Human Monocyte Nucleofector buffer (V4XP-3024; Lonza) and the Lonza 4D-Nucleofector platform were used according to the manufacturer’s instructions with the human monocyte nucleofection program. The nucleofected monocytes were cultured in RPMI 1640 medium (Corning) supplemented with 10% (vol/vol) FBS (Gibco) and human recombinant M-CSF (20 ng/ml; PeproTech) for 48 h before the following experiments.

Zhang B., Zhang Y., Xiong L., Li Y., Zhang Y., Zhao J., Jiang H., Li C., Liu Y., Liu X., Liu H., Ping Y.F., Zhang Q.C., Zhang Z., Bian X.W., Zhao Y, & Hu X. (2022). CD127 imprints functional heterogeneity to diversify monocyte responses in inflammatory diseases. The Journal of Experimental Medicine, 219(2), e20211191.

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Monocyte Nucleofection of siRNA

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For short interfering RNA (siRNA) experiments, 10⁷ human CD14⁺ cells were nucleofected with 0.32 nmol of siRNA oligonucleotides using a Nucleofector kit (Lonza, Basel, Switzerland) as previously described.^{48 (link)} “Human Monocyte Nucleofector buffer (Lonza) and the AMAXA Nucleofector System program Y001 for human monocytes were used according to the manufacturer’s instructions. We tested two different sets of siRNAs and MEF2C-specific (#4392420 ID8652 for MEF2C #1 and ID 8653 for MEF2C #2) and control (#4390843) siRNAs were obtained from Thomas Fisher Scientific”.^{25 (link)}

Fujii T., Murata K., Mun S.H., Bae S., Lee Y.J., Pannellini T., Kang K., Oliver D., Park-Min K.H, & Ivashkiv L.B. (2021). MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS. Bone Research, 9, 4.

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Transfecting Human Monocytes with siRNA

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For RNA interference (RNAi) experiments, primary human monocytes (10 × 10⁶ cells) were transfected
with 0.2–0.4 nmol of siRNA oligonucleotides (listed in the key resource table)
using a Nucleofector kit. Human Monocyte Nucleofector buffer and the AMAXA Nucleofector System (Lonza) program Y001 for human
monocytes were used according to the manufacturer’s instructions.

Kusnadi A., Park S.H., Yuan R., Pannellini T., Giannopoulou E., Oliver D., Lu T., Park-Min K.H, & Ivashkiv L.B. (2019). The cytokine TNF promotes transcription factor SREBP activity and binding to inflammatory genes to activate macrophages and limit tissue repair. Immunity, 51(2), 241-257.e9.

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Monocyte Knockdown Using siRNA

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Immediately after isolation, primary human monocytes were nucleofected with ON-TARGET plus SMARTpool short interfering RNAs (siRNA) specific for MAF purchased from Dharmacon. ON-TARGETplus Non-targeting Pool was used as control. An additional control siRNA and two additional MAF-targeting siRNAs were used with comparable results. Human Monocyte Nucleofector buffer (Lonza Cologne) and the AMAXA Nucleofector System program Y001 for human monocytes were used according to the manufacturer’s instructions.

Kang K., Park S.H., Chen J., Qiao Y., Giannopoulou E., Berg K., Hanidu A., Li J., Nabozny G., Kang K., Park-Min K.H, & Ivashkiv L.B. (2017). Interferon-γ Represses M2 Gene Expression in Human Macrophages by Disassembling Enhancers Bound by the Transcription Factor MAF. Immunity, 47(2), 235-250.e4.

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Monocyte Knockdown via Nucleofection

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Immediately after isolation, primary human monocytes were nucleofected with On-Target plus SMARTpool short interfering RNAs (siRNA) purchased from Dharmacon Inc. (Lafayette, Colorado, USA) specific for MNK1, MNK2 or MYC. Non-targeting siRNA#5 was used as control. Human Monocyte Nucleofector buffer (Lonza Cologne, Cologne, Germany) and the AMAXA Nucleofector System program Y001 for human monocytes were used according to the manufacturer's instructions.

Su X., Yu Y., Zhong Y., Giannopoulou E.G., Hu X., Liu H., Cross J.R., Rätsch G., Rice C.M, & Ivashkiv L.B. (2015). Interferon-γ regulates cellular metabolism and mRNA translation to potentiate macrophage activation. Nature immunology, 16(8), 838-849.

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Knockdown of IL10 eRNAs using LNAs

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LNAs (LNA^TM longRNA GapmeR) were designed and synthesized by Exiqon. Knockdown experiments with LNAs were performed using Human Monocyte Nucleofector buffer (Lonza Cologne) and the AMAXA Nucleofector System program Y001 for human monocytes according to the manufacturer’s instructions. Two different LNA^TM longRNA GapmeRs (negative control A and B) were used as control. Cells were harvested for RNA analysis 24 h after transfection. The following LNAs were used for knockdown studies of IL10 eRNAs. Custom LNA for IL10 eRNA (HSS-16): ATAGAGAGGAGATGCA, GCAGTCTAGCTTGGTG. Custom LNA for IL10 eRNA (HSS + 6): GGATTTGGCGGGAGTT, TCCTAGTGCCAGAAGC.

Kang K., Bachu M., Park S.H., Kang K., Bae S., Park-Min K.H, & Ivashkiv L.B. (2019). IFN-γ selectively suppresses a subset of TLR4-activated genes and enhancers to potentiate macrophage activation. Nature Communications, 10, 3320.

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Monocyte Knockdown via Nucleofection

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Monocyte Knockdown Using siRNA Nucleofection

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Freshly-isolated PBM were resuspended in human monocyte-nucleofector buffer (Lonza, USA) and mixed with SMARTPool CD32b (FcγRIIb), CD31 siRNA or Scr control (Dharmacon, GE Life Sciences, CO) at a concentration of 200 nM. Cells were nucleofected using the Amaxa Nucleofector I (Lonza, USA), and cultured for 72 hours in complete RPMI supplemented with 100 μg/mL of recombinant human M-CSF (R&D Systems, MN). Knockdown was confirmed by western blotting as described above.

Merchand-Reyes G., Robledo-Avila F.H., Buteyn N.J., Gautam S., Santhanam R., Fatehchand K., Mo X., Partida-Sanchez S., Butchar J.P, & Tridandapani S. (2019). CD31 acts as a checkpoint molecule and is modulated by FcγR-mediated signaling in monocytes. Journal of immunology (Baltimore, Md. : 1950), 203(12), 3216-3224.

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Nucleofection of Human Macrophages for RNAi

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For RNA interference (RNAi) experiments, primary human macrophages (10⁷ cells) were nucleofected with 0.2 –0.4 nmol of siRNA oligonucleotides using a Nucleofector kit (Lonza) as previously described (Park-Min et al., 2014 ). Human Monocyte Nucleofector buffer (Lonza Cologne) and the AMAXA Nucleofector System program Y001 for human monocytes were used according to the manufacturer’s instructions. We tested three different sets of siRNAs or antisense LNAs, using at least three donors unless otherwise stated. COMMD1-, and E2F1-specific and control siRNAs were obtained from Thomas Fisher Scientific. COMMD1- and E2F1- specific and control antisense LNAs were purchased from Exiqon. Sequences are shown in Table S1.

Murata K., Fang C., Terao C., Giannopoulou E.G., Lee Y.J., Lee M.J., Mun S.H., Bae S., Qiao Y., Yuan R., Furu M., Ito H., Ohmura K., Matsuda S., Mimori T., Matsuda F., Park-Min K.H, & Ivashkiv L.B. (2017). Hypoxia-sensitive COMMD1 Integrates Signaling and Cellular Metabolism in Human Macrophages and Suppresses Osteoclastogenesis. Immunity, 47(1), 66-79.e5.

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