Hi all!
Does anyone have a good protocol for efficient transfection of plasmids into RAW 264.1 macrophages?
Cheers!
Olivier
Here's an electroporation kit for RAW 264.7 (https://altogen.com/product/raw-264-7-electroporation-kit-lymphoma-cells-tib-71/). Generally speaking suspension cell lines are better electroporated than lipofected, mainly because electroporation will guarantee that all of the cells will be affected (with the electric field introducing pores in the cell membranes). As long as you have a sufficient concentration and distribution of your complexes, you should have good efficiency.
Hi Olivier,
You can use JetPEI or Amaxa, have a look at the protocols in Kasmapour et al. 2012 or Pei et al. 2014. I am happy to provide more details of course.
Best wishes, Max
You will usually find a protocol depending on the transfection reagent that you use.
We have used two different reagents, lipofectamine and Fugene-HD. I would recommend Fugene-HD as it gives better efficiency and low cell death. There is a newer version of lipofectamine available which is similar to Fugene-HD. Below is the protocol for Fugene-HD for MH-S alveolar macrophage cell line. The same is true for Raw's. Note that this is optimized for a 35 mm dish. If you are well with smaller area, you will have to change the DNA amount and the transfection reagent (Refer to manufacturers protocol about these ratios for optimization)
1) Briefly, 1x105 cells/cm2 macrophages were plated in 35 mm Willco (WillCo Wells BV, Amsterdam, The Netherlands) or ibidi GmbH (München, Germany) glass bottom dish in 1ml of RPMI-1640 complete media at 37°C with 5% CO2 and allowed to adhere overnight.
2) The next day, media was changed to 875 µl of fresh RPMI-1640 complete media
3) Mix 2 micrograms of DNA with 100 µl RPMI media only containing 10mM HEPES in a 5 ml sterile polystyrene tube.
4) Add 6 µl of room temperature FuGENE HD transfection reagent to the center of the tube in step 3, being careful to only allow the FuGENE HD to mix with the DNA-media complex and not directly touch the tube.
5) Incubate for 15 minutes at room temperature
6) Add the contents of the tube to cells in a drop-wise fashion
7) Allow cells to incubate at 37°C with 5% CO2 overnight
8) After 10-12 hours replace with fresh medium
Good luck,
Gaurav.
Hi Olivier,
check out this paper, we published some year ago...
A+, Jp
Hi Olivier,
We used to do it with amaxa (they provide a specific protocol) and it worked for plasmids and shRNA, maybe you can check with Christel Verollet for tips and advices if you go this way.
Cheers,
Guillaume.
Article HIV-1 Nef Triggers Macrophage Fusion in a p61Hck-and Proteas...
We used Amaxa yesterday. Plenty fluorescent Raw264 cells today (>50%). However, many dead cells as well. We used Y-01 program with nucleofectior I.
Hey,
I used K2 reagent 2 month ago for RAW cells and it worked fine with me. I had a transfection rate of around 50%.
http://www.biontex.com/con_4_6_4/cms/front_content.php?changelang=1&idcat=260
all the best,
Sebastian
Oliver,
The only method of transfection I use is Amaxa. Favorite program is U-017. This is a very harsh program. If you are using only the adherent cells, I recommend this as you can put up to 1.5 million cells in the electroporation cuvette at once to meet any density requirements. When transfecting a new cell line, always run at least 5 different programs with only the pmax supplied with the Amaxa kit and see which one is more conducive to your project as far as death rate, transfection percentage and density. Hope this helps.
Matt
Dear Olivier,
I would be very glad sending you samples of some of our transfection that gave some good results in Raw264.7 cells;
if you want to give a try, please do not hesitate to contact me at: [email protected] ;
good luck with your experiment,
best regards,
Cedric
Here's an electroporation kit for RAW 264.7 (https://altogen.com/product/raw-264-7-electroporation-kit-lymphoma-cells-tib-71/). Generally speaking suspension cell lines are better electroporated than lipofected, mainly because electroporation will guarantee that all of the cells will be affected (with the electric field introducing pores in the cell membranes). As long as you have a sufficient concentration and distribution of your complexes, you should have good efficiency.
Macrophages that have not engulfed anything have great reserves of plasma membrane which makes transfection with lipid reagents difficult. Electroporation is the best way to go.
Matt
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