Dear Dora,

Here are my suggestions for infection of special cell lines with lentivirus, such as MEF.

1. Suspension cells

We recommend using flat fillet centrifuging transfection to infect suspension cells or semi-suspension cells. Add virus suspension into cell culture dish, sealing tightly, and centrifuge at low speed of 200×g for 1 hour in the flat fillet centrifuge. Place cells in cell culture incubator after centrifuging transfection. If the flat fillet centrifuge is inaccessible, you can suspend the cells and transfer cells into centrifuge tubes, followed by low-speed centrifuge, and discard the most of supernatant. Add virus suspension into the tubes, resuspending cells, place it at room temperature for 15 min (no more than 30 min), and transfer the cells and virus suspension into plate to culture. Replace with fresh culture medium the next day.

2. For cells difficult to infect, like DC cells, we recommend repeated infections. Replace with fresh virus suspension 24 hours after the first infection. Repeated infections can increase the infection efficiency markedly.

3. Non-dividing primary cells

We recommend high-titer adenovirus to infect these cells like BMSC.

Genemedi got a rich experience in lentivirus production and construction of stable cell lines, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.

I agree MEFs are difficult to transfect, so you might have to use a lentivirus approach if you want to work with MEFs. This paper claims mouse myeloma lines NSO and SP2/0 are "easy to transfect": http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-62703-586-6_13

In reality, though, different transfection reagents work well with specific cell lines. There are transfection reagents adapted to specific murine cell lines that you can find commercially available:

C2C12 cells: http://signagen.com/pre-optimized-transfection-reagents--c-135_139/genjet-in-vitro-dna-tranfection-reagent-for-c2c12-cells

B16-F10 cells: http://signagen.com/pre-optimized-transfection-reagents--c-135_139/genjet-in-vitro-dna-tranfection-reagent-for-b16-f10-cells

Hi Dora,

I compared MEF, NIH 3T3 and L929 cells with a lipofectamine 2000 protocol. I had nice results (up to 60%- of transfection efficiency using NIH-3T3. You have to set-up the plasmid quantity and lipofectamine concentration. I used 200ng of plasmid and 2µL lipofectamine /well (12 well plate). Hope it will help,

Sophie

Hi Dora,

you can even try Lipofectamine LTX with Plus reagent which can be used also for

primary cells, such as fibroblasts. But I agree with Mark that it is better to use lentivirus approaches with MEFs, that works quite well.

Hi

I am using Fugene HD transfection reagent for primary MEFs. Its working fine for me. you could try it. Here is link

http://fi.promega.com/products/reporter-assays-and-transfection/transfection-reagents/fugene-hd-transfection-reagent/

Hi,

C2C12 or N1E-115 cells with Lipofectamine 2000. You should test several conditions.

Jorge

Hi Dora,

For MEF transfetion I would highly suggest you trying DreamFect and DreamFect Gold transfection reagents.

Both transfection reagents are really efficient with multiple cell lines and they were shown to transfect MEF with good level. For example you can refer to the following publications : Kríz V et al, J Biol Chem, 2013 (http://www.ncbi.nlm.nih.gov/pubmed/24265322); Keil E et al, Cell Death Differ, 2013 (http://www.ncbi.nlm.nih.gov/pubmed/23059785); or Agarwal NK et al, Oncogene, 2013 (http://www.ncbi.nlm.nih.gov/pubmed/22777355).

Do not hesitate to contact me at [email protected] if you want more information or samples to test,

have a nice day and good luck for your following experiments,

Cedric

Thank you all very much for the help, I managed to troubleshoot my issue with the NIH 3T3 cells.

Have a nice day!!!!

Hi Sophie

What was  the transaction efficiency of MEF cell line with lipfectamine 2000?

Dear Dora,

Here are my suggestions for infection of special cell lines with lentivirus, such as MEF.

1. Suspension cells

We recommend using flat fillet centrifuging transfection to infect suspension cells or semi-suspension cells. Add virus suspension into cell culture dish, sealing tightly, and centrifuge at low speed of 200×g for 1 hour in the flat fillet centrifuge. Place cells in cell culture incubator after centrifuging transfection. If the flat fillet centrifuge is inaccessible, you can suspend the cells and transfer cells into centrifuge tubes, followed by low-speed centrifuge, and discard the most of supernatant. Add virus suspension into the tubes, resuspending cells, place it at room temperature for 15 min (no more than 30 min), and transfer the cells and virus suspension into plate to culture. Replace with fresh culture medium the next day.

2. For cells difficult to infect, like DC cells, we recommend repeated infections. Replace with fresh virus suspension 24 hours after the first infection. Repeated infections can increase the infection efficiency markedly.

3. Non-dividing primary cells

We recommend high-titer adenovirus to infect these cells like BMSC.

Genemedi got a rich experience in lentivirus production and construction of stable cell lines, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.