I am trying to transfect THP-1 cell line using with lipofectamine. But it does not work. I want to try lipofectamine before electroporation. Can you give me your protocol ? If the lipofectamine does not work I also need your electroporation protocol.
Thank u for your help.
Hi,
Lipofectamine 2000 is sensitive to changes in temperature. please make sure to store it at 4C and avoid freeze/thaw cycles at all cost.
I have attached the transfection protocol in primary cortical cultures that I use for my work. I hope this will help resolve your problem.
Hi
Thermo fisher has Lipofectamine® LTX Reagent,which is suitable for your experiment.
below is the link provide information about your work.
https://www.thermofisher.com/in/en/home/references/protocols/cell-culture/transfection-protocol/transfecting-plasmid-dna-into-thp-1-cells-using-lipofectamine-ltx-reagent.html
Hope this helps
Electroporation Protocol ( please see
https://www.neb.com/protocols/1/01/01/electroporation-protocol-c2986)
1.Prepare 17mm x 100mm round-bottom culture tubes (e.g. VWR #60818-667) at room temperature. Place SOC recovery medium in a 37°C water bath. Pre-warm selective plates at 37°C for 1 hour.
2.Place electroporation cuvettes (1mm) and microcentrifuge tubes on ice.
3.As a positive control for transformation, dilute the control pUC19 by 1:5 to a final concentration of 10 pg/μl using sterile water. Heat-denatured ligation reactions can be used for electroporation directly; however, column purification is recommended.
4.Thaw NEB Turbo Electrocompetent cells on ice (about 10 min) and mix cells by flicking gently. Transfer 25 μl of the cells (or the amount specified for the cuvettes) to a chilled microcentrifuge tube. Add 1 μl of the DNA solution.
5.Carefully transfer the cell/DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette. Electroporate using the following conditions for BTX ECM 630 and Bio-Rad GenePulser electroporators: 2.1 kV,
100 Ω, and 25 μF. The typical time constant is ~2.6 milliseconds.
6.Immediately add 975 µl of 37°C SOC to the cuvette, gently mix up and down twice, then transfer to the 17mm x 100mm round-bottom culture tube.
7.Shake vigorously (250 rpm) or rotate at 37°C for 1 hour.
8.Dilute the cells as appropriate then spread 100-200 μl cells onto a pre-warmed selective plate.
9.Incubate plates 8 hours to overnight at 37°C.
Hello
If you want to transfect siRNA, do not hesitate and try Viromer BLUE or GREEN.
For plasmid transfection, you can reach 25% with Viromer RED, but using mRNA (produced by IVT or provided by a supplier) will give much more chance to express your protein.
Have a look to users' feedback on the attached pdf, and please contact me if you want to test our technology.
Bests,
Sandra
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