How much Lipofectamine RNAimax shoiuld be used for siRNA transfection?

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Looking for a protocol for isolation of nuclei from mammalian cells?

I am trying to get pure cytoplasmic and nuclear fractions from mammalian cells after infection, for further viral DNA quantification. After fractionation and DNA extraction, I quantified...

09 October 2017 3,367 4 View

Hello, I need to generate stable cell lines expressing number of nucleoporins fused to GFP, Does anyone have experience on how to generate them?

Each cell line would express one of the nucleoporins fused to GFP.

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No title

02 March 2021 3,060 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

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Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

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Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

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What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View

Dr. Shilpa Thakur

Hi,

You can use 3-4uL lipofectamine per well. Perform transfection in opti-MEM without serum and you can go lower the cell confluency as well (60-80%)

Karthikeyan Krishnamurthy

Go with low cell density (seed the cells 24 hours before transfection) if you want best result. Use medium without PS, Keep your cells with low medium overnight (like 0.5ml) them increase the medium next day.

Vikrant Mehta

About 4ul of lipofectamine would be enough. By the way Krishnamurthy's comment suggesting transfection at low cell density is worth trying . See the attached PDF for more details

Mahmoud Farghali

Thank you Dr. Shilpa Thakur . I am trying now different conditions as lower confluency or more lipofectamine, Thanks. @ Dr. Shilpa Thakur

@ Arian Rahimi

Thank you. I will try different conditions of confluency and lipofectamine RNAimax , if it doesn't work, I will try the reagent you mentioned. Thanks

@ Katerina Kapodistria

Thanks Katerina. I am trying now lower cells conflency (from 30,000-100,000) cells/well in 24 well-plate. If it doesn't work, I will try the conditions you suggested. Thanks

@ Karthikeyan Krishnamurthy

I am trying a range of lipofectamine amounts along with a range of cell density. Thanks for you suggestions.

@ Vikrant Mehta

Thanks Vikrant. Yes, I think lowering cell density with increasing Lipofectamine may solve the issue.

Ted Bremmer

HepG2 is known to be pretty picky with regards to transfection conditions - experimenting with different concentrations (of both cells and reagents) can help, but also trying different chemical techniques/additives (see https://altogen.com/product/hepg2-transfection-reagent-hepatocellular-carcinoma/). There are some ways of improving transfection that involve better penetration capacities of liposomes (through conjugation with other organic compounds) and it can be worth looking into other kits for higher efficiencies.