Dear Ariel, I have mentioned this previously. We have received good results using nucleofection for mature THP-1 cells. Although we have not systematically investigated the THP-1 protocol for using primary human monocyte-derived macrophages, we received first good results using the THP-1 protocol also for the primary cells. One bottleneck of our protocol for using primary cells is that macrophages become more difficult to dettach using Accutase with progressing maturation. However, I think that our THP-1 protocol is worth a try for primary macrophages as long as you have the equipment available.

Our protocols have been published:

Improved protocol for efficient nonviral transfection of premature THP-1 macrophages.

Maess MB, Buers I, Robenek H, Lorkowski S.

Cold Spring Harb Protoc. 2011; 2011(5):pdb.prot5612.

doi: 10.1101/pdb.prot5612.

PMID: 21536764

Efficient non-viral transfection of THP-1 cells.

Schnoor M, Buers I, Sietmann A, Brodde MF, Hofnagel O, Robenek H, Lorkowski S.

J Immunol Methods 2009; 344(2):109-15.

PMID: 19345690

Please let me know your email adress if you do not have acces to the papers. I can send you PDF reprints as well as a step-by-step protocol.

Best regards,

Stefan

Thank you, I read about the commercial kits to improve the efficiency of transfections. I want to visualize the translocation of fluorescent labeled HDAC due to my treatments.

We haven't had success with plasmid transfections, but the Lonza Nucleofector works well for siRNA. http://www.lonza.com/products-services/bio-research/transfection/nucleofector-kits-for-primary-cells/nucleofector-kits-for-primary-blood-cells/nucleofector-kits-for-mouse-macrophages.aspx

Macrophages are notoriously hard to transfect for 2 (possible) reasons. 1) They are highly differentiated - so the machinery are not geared towards overexpressing plasmids. 2) They are macrophages so they eat foreign things, incl. plasmids.

Some have tried lentivirus with some success. Electroporation might work - but Amaxa's system has little flexibility. Perhaps a Biorad electroporation with the extra capacitor box (quite expensive) might work - as you can change the settings.

DNA ought to be prepared using an "Endo-free" kit (Sigma or Qiagen make them). The vectors themselves would not 'activate' macrophages, but the Endotoxins (e.g. LPS) from E.coli would.

I would suggest before launching into Primary macrophages, try transfecting RAW264.7 macrophage-like cell line - they are a bit easier and that would build up some confidence. All the best!

We routinely transfect primary human macrophages with the Neon transfection system (Invitrogen/Life Technologies). It gives similar transfection efficiencies compared to Lonza (up to 100% for siRNA, 10-15% for vectors), but is much superior in terms of cell viability and perservation of normal morphology. Plus, you only have one kit for all cell systems and are able to adjust parameters such as pulse number or length by yourself.

Dear Ariel, I have mentioned this previously. We have received good results using nucleofection for mature THP-1 cells. Although we have not systematically investigated the THP-1 protocol for using primary human monocyte-derived macrophages, we received first good results using the THP-1 protocol also for the primary cells. One bottleneck of our protocol for using primary cells is that macrophages become more difficult to dettach using Accutase with progressing maturation. However, I think that our THP-1 protocol is worth a try for primary macrophages as long as you have the equipment available.

Our protocols have been published:

Improved protocol for efficient nonviral transfection of premature THP-1 macrophages.

Maess MB, Buers I, Robenek H, Lorkowski S.

Cold Spring Harb Protoc. 2011; 2011(5):pdb.prot5612.

doi: 10.1101/pdb.prot5612.

PMID: 21536764

Efficient non-viral transfection of THP-1 cells.

Schnoor M, Buers I, Sietmann A, Brodde MF, Hofnagel O, Robenek H, Lorkowski S.

J Immunol Methods 2009; 344(2):109-15.

PMID: 19345690

Please let me know your email adress if you do not have acces to the papers. I can send you PDF reprints as well as a step-by-step protocol.

Best regards,

Stefan

Dear Stefan: Can you send me via email these protocols? Thank you. [email protected]

Thank you all for your answers. They are extremely helpful. Stefan Linder, how did you measure the efficiency of siRNA? And vectors for that matter?

And Stefan Lorkowski, I was able to get the papers and protocol through my university's library. Thank you so much for your help!

Dear Ariel,

I´m glad that all the answers were helpful. We measured siRNA transfection efficiency on the basis of a fluorescently labeled control siRNA. For vectors, we use GFP- or mCherry-labeled constructs and determined the ratio of fluorescent cells.

For a short protocol, here is an excerpt from a recent paper of ours (Wiesner et al., JCS, 2013): Transient transfections were performed using the Neon® Transfection System (Life technologies, Invitrogen, Darmstadt, Germany) with standard settings (1000 V, 40 ms, 2 pulses) and appropriate amounts of siRNA (650 ng) and plasmids (0.5 -1.5 µg / 10 000 cells), respectively.

All the best

Stefan

Dear Stefan Lorkowski,

I do have the same problem as Ariel Burns. I have tried to check your protocol/papers but i cannot have access to it. Could you please send it to my email address? [email protected]

Thank you so much!

Bea

With RAW 264.7, a macrophage cell line, it is best to use electroporation, and we've had good results using the technique (see https://altogen.com/product/raw-264-7-electroporation-kit-lymphoma-cells-tib-71/). Primary macrophages are very difficult to transfect, but if you do some optimization runs beforehand you should get sufficiently good results for your research.

Dear Dr. Lorkowski

May you send me your papers and improved protocol about non-viral transfection

of premature THP-1 macrophages. We tried to transfect cow monocyte-derived macrophage with DNA vectors using Lipofectamine 3000 or PromoFectine unsuccessfully.These two transfectant works in the same cells with siRNA.

Thanks in advance for your collaboration