I saw this and thought it may help,

Lipid-Mediated Transfection of Human Astrocytes

https://www.thermofisher.com/us/en/home/references/protocols/neurobiology/neurobiology-protocols/lipid-mediated-transfection-of-human-astrocytes.html

Transfecting plasmid DNA into human astrocytes using Lipofectamine® LTX Reagent Use this procedure to transfect plasmid DNA into Gibco® Human Astrocytes using the Lipofectamine® LTX Reagent in a 24-well format (for other formats, see Scaling up or down transfections, below). All amounts and volumes are given on a per well basis.

1.The day before transfection, prepare Human Astrocytes that have recovered from cryopreservation and have reached 80% confluency. Use StemPro® Accutase® to detach the cells and count the cells. Plate 5 × 104 cells per well in 0.5 mL of complete growth medium. Cell density should be 80–90% confluent on the day of transfection. 2.For each well of cells to be transfected, dilute 0.5 μg of DNA into 100 μL of Opti-MEM® I Reduced Serum Medium without serum. 3.Using PLUS™ Reagent: Mix PLUS™ Reagent gently before use, then add 0.5 μL PLUS™ Reagent (a 1:1 ratio to DNA) directly to the diluted DNA. Mix gently and incubate for 5–15 minutes at room temperature.

4.For each well of cells, dilute 1.5–3.0 μL of Lipofectamine® LTX into the above diluted DNA solution, mix gently and incubate for 25 minutes at room temperature to form DNA-Lipofectamine® LTX complexes.

5.Remove growth medium from cells and replace with 0.5 mL of complete growth medium. Add 100 μL of the DNA-Lipofectamine® LTX complexes directly to each well containing cells and mix gently by rocking the plate back and forth.

6.Complexes do not have to be removed following transfection. Incubate the cells at 37°C in a CO2 incubator for 18–24 hours post-transfection before assaying for transgene expression.

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I have no experience with astrocytes, but you really ought to transfect in the minimal media and change the media after several hours (lipofection is usually complete within a few hours). In terms of your dose, it sounds appropriate to me, although I work on primary epithelial cultures (which are much more robust).

You can also try transfecting in less media, which allows you to use less Lipofectamine and RNAi reagents, and usually turns out to be less toxic to the cells.

Lastly, have you performed a transfection efficiency protocol, using something like FAM? It often allows you to quantify the least amount of transfection reagent necessary for a high transfection efficiency (as opposed to using the manufacturer-recommended "bucketload").

Hope some of this helps,

Dennis

I saw this and thought it may help,

Lipid-Mediated Transfection of Human Astrocytes

https://www.thermofisher.com/us/en/home/references/protocols/neurobiology/neurobiology-protocols/lipid-mediated-transfection-of-human-astrocytes.html

Transfecting plasmid DNA into human astrocytes using Lipofectamine® LTX Reagent Use this procedure to transfect plasmid DNA into Gibco® Human Astrocytes using the Lipofectamine® LTX Reagent in a 24-well format (for other formats, see Scaling up or down transfections, below). All amounts and volumes are given on a per well basis.

1.The day before transfection, prepare Human Astrocytes that have recovered from cryopreservation and have reached 80% confluency. Use StemPro® Accutase® to detach the cells and count the cells. Plate 5 × 104 cells per well in 0.5 mL of complete growth medium. Cell density should be 80–90% confluent on the day of transfection. 2.For each well of cells to be transfected, dilute 0.5 μg of DNA into 100 μL of Opti-MEM® I Reduced Serum Medium without serum. 3.Using PLUS™ Reagent: Mix PLUS™ Reagent gently before use, then add 0.5 μL PLUS™ Reagent (a 1:1 ratio to DNA) directly to the diluted DNA. Mix gently and incubate for 5–15 minutes at room temperature.

4.For each well of cells, dilute 1.5–3.0 μL of Lipofectamine® LTX into the above diluted DNA solution, mix gently and incubate for 25 minutes at room temperature to form DNA-Lipofectamine® LTX complexes.

5.Remove growth medium from cells and replace with 0.5 mL of complete growth medium. Add 100 μL of the DNA-Lipofectamine® LTX complexes directly to each well containing cells and mix gently by rocking the plate back and forth.

6.Complexes do not have to be removed following transfection. Incubate the cells at 37°C in a CO2 incubator for 18–24 hours post-transfection before assaying for transgene expression.

The Comparative Utility of Viromer RED and Lipofectamine for Transient Gene Introduction into Glial Cells

Article The Comparative Utility of Viromer RED and Lipofectamine for...

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We did transfect a glioma (astrocyte) cell line with a YFP-tagged construct of our protein of interest using Lipofectamine 2000. With all our best efforts the tranfection efficiency was around 20-22%, again for the protein we were studying. In our experience, it is not the quantity of lipofectamine, but the concentration and quality of the DNA/plasmid that you are transfecting, which proves to be lethal.

You need to play around and titrate the optimal dose of DNA that gives minimal cell death. One should also consider the quality of your plasmid prep to ensure that you don't have any endotoxins left over from the bacterial source. Hope this helps. If you have any questions, I would be happy to answer.

Thanks everyone for your help!

Raj, our limiting factor is the number of cells (embryonic astrocytes) so, a titration maybe will delay us too much. Which is the concentration you use? We used 500 ng DNA/well, is the one recommended in the protocol. Thank you very much again.

Hi everyone!

We have repeated the experiment this week and changing a few things the results are much better.

In the first place, we changed the growing media by Optimem (without antibiotics) 30'-1h before. In the second place, we left the Optimem with the transfection complex overnight and the next morning we changed it by growing media. The concentration of Lipofectamine was the minimum recommended and the amount of DNA was 500 ng per well (24 well plate).

I attached some images of the results.

Thanks everyone for your recommendations!

Hi Rucsanda,

I'm happy to hear that you managed to transfect your astrocytes with such great efficiency. Sorry for asking, but I'm about to transfect astrocytes myself and would like some advice if you have.

So you used Lipofectamine 3000, incubated complexes ON at RT and added to your cells in growth medium without antibiotics but with serum (medium of the cells was changed 1 h prior to transfection)?

What would you estimate the efficiency to be, 20 %?

Thank you in advance.

Rucsanda Pinteac. Have you seen my questions? :)

Frederik Ravnkilde Marlet sorry for the delay, I haven't seen your questions until today. We performed the transfection in OPTIMEM without antibiotics and without serum. Then, we changed the OPTIMEM for growing medium with all the components. We didn't incubate the complexes (DNA-Lipofectamine) overnight, we followed the Lipofectamine 3000 protocol. We incubated our cells with the complexes ON at 37ºC. The next morning we changed the Optimem for the same growing media we were using to grow the astrocytes.Hope this helps and sorry for the delay.