Dear Muhammad,

In my opinion, you can take lentivirus-mediated shRNA knock-down into your consideration. Since it is difficult for suspension cells to be transfected using transfection reagents. Lentiviruses mediate integration of exogenous genes into host genome, and stable and long-term expression of exogenous genes, which may meet your demands.

Here is our transfection protocol with lentivirus in 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 200g 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.

Genemedi got a rich experience in virus production and transfection, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.

Hi Imran,

The protocol for transfecting suspended cells is very similar to adherent cells other than they generally do not need to be plated the day before. Most transfection reagents suggest using antibiotic free medium.

I transfect THP-1 cells (monocytic cells) with lipofectamine LTX reagent (using the PLUS reagent). There are different protocols for using lipofectamine with specific cell lines. Here is one for HL-60 cells: www.lifetechnologies.com/uk/en/home/references/protocols/cell-culture/transfection-protocol/transfecting-plasmid-dna-into-hl-60-cells-using-lipofectamine-ltx-reagent.html.

I have also used MACSfectin with some succeshttps://www.miltenyibiotec.com/en/products-and-services/macsmolecular/transfection-and-transduction/macsfectin-reagent.aspx

Otherwise, more harsh techniques, such as electroporation, may result in better transfection efficiencies (especially with hard to transfect cell lines like HL-60 and THP-1) but with the risk of losing cell viability.

Each cell line is very different with regards to ease of transfection and I have not used HL-60. Also, I have not transfected siRNA into suspension cells. However, if THP-1 are to go by, it is do-able. Just be patient and make sure you are willing to spend time optimising the assay.

Good luck!

José

I have tried Lipofectamine RNAiMax and FuGENE HD to knockdown some genes in THP-1 (monocytes) and they both worked great. I was able to get 60-70% transfection efficiency within the first 24-h and very high viability (>90%).

I would say Lipofectamine RNAiMax is easier to use (since I use stealth siRNA from the same company). FYI, for suspension cells, I would prefer to do reverse transfection (you seed the cells and transfect them on the same day). I have never worked with HL-60 before, and not sure about your siRNA, but you can always do some optimization by testing different siRNA:Liposome ratio.

Did you try Lipofectamine2000? All Invitrogen Lipofection reagents work well.

Try ScreenFect®A if you are having trouble with transfection of your suspension cells with siRNA/miRNA.

Take a look at: http://www.sciencedirect.com/science/article/pii/S016816561300374X

I've heard of a new one in the market called Happyfect. See if you can get a sample from the company (Mayflower, in STL) and try it out. Good luck.

One customer reports success with Jurkat cells and HappyFect-RNAi. Non-toxic, so can also try serial transfection 2 hours after the first. Not sure who distributes in South Korea, so go direct to manufacturer in UK, www.tecrea.co.uk

Thank you all for sharing your knowledge...Transfection in suspension cells is of course difficult and so far I haven't tried it, but based on your experiences I can simply conclude that we have a window to look through...

I am interested to knock down Btg2 (B-Cell translocation gene 2) in HL-60 cells. I guess transfection in THP-1 cells is easier than HL-60..... I have Fugene and Lipofectamine 2000 in hand, so will give them a try. If these reagents did not work then the suggested options are lipofectamine LTX, ScreenFect®A, Happyfect (HappyFect-RNAi)....Thank you once again...

Dear Imran!

I am right now struggling with a similar problem, but in my case it is about transfecting lymphoma cell lines with siRNA. Anyway, there is a paper in BioTechniques by Basiouni et al 2012 using HL-60 cells that by egg white(!) are manipulated to attach down and then become easy to transfectb according to the authors. Could be worth trying :-)

Good luck

If you need to use viral technologies for knockdown then look up this company who has some very good reagents and services. www.sirion-biotech.com/

You could try Lipofectamine2000 or FuGene HD, both of these two reagents should be able to transfect suspension cells.

@ Thomas Stromberg...The information is interesting and also the protocol is simple but we might face too problems; (1) High risk of contamination (2) Coating plates with the egg white might induce signaling in the cells, just like fibronectin coating. I give you one more information, if you suspend any suspension culture in serum free media (with out any coating), with in minutes the cells are attached to the culture dish, however, they do detach after several hours and in fact this rate of detachment is cells context dependent. But whether such type of adherence can increase transfection efficiency, this is something I haven't tested yet and importantly suspending cells in serum free media activates NF-kB pathway and my gene of interest is a confirmed target for NF-kB. Any ways it would be nice to try egg coated plates for transfection. Thanks

@Umesh Patel ...Thanks for information....Actually I have siRNA sequences which are working perfectly in adherent cells that's why I want to transfect the same siRNA in suspension cells. I understand lentiviral technology is the absolute solution to this problem and if I failed to do it the other way then of course I will think about viral transduction.

@ Yuning Hou and Ömer Bayrak...Thanks for information..... Fugene HD and Lipofectamine 2000 are the available reagents that I am going to use first.....and then the other strategies...Thank you all once gain....

I have tried Lipofectamine 2000(invitrogen) both for adherent and suspension cells. It worked well. Just centrifuge cells in all steps.

Panagiotis Apostolou ...thanks....what type of suspension cells did you use?

Hello! Dear though I have not worked on these type of cells yet I would agree with Dr. Panagiotis and recommend you to try Lipofectamine 2000 (Invitrogen). We have used this reagent on mammalian mesenchymal stem cells which are also very hard to transfect. We have found this very successful in ratios of 1:1 with DNA for 24 hours. I hope it will help you. I am sending herewith the broucher and article for its use.

Regards.

I think electroporation is better for suspension cells.

Both from commercial cell lines (all cancer types) as well as cell lines derived from patients's CTCs.

We used SaintRed from Synvolux and had efficient knock-down in K562 cells, which also dont like transfections in general. This reagent gave good results in numerous adherent cells, too.See paper below. Only, whenever you receive a new batch of this reagent, you may want to briefly reassess toxicity, as this varies somewhat from lot to lot.

http://www.ncbi.nlm.nih.gov/pubmed/21368884

hi,

hyperfect from Qiagen company works very well for siRNA transfection of suspension cells. 100 nM final concentrated siRNA and 6 microliters of Hyperfect is enough. i have silenced my target gene in K-562 cells for a duration of 12 days with only 1 transfection..but i have to say that it is not as easy to transfect HL-60 cells as K-562; since HL-60 cells are smaller..

so initially, i advice u to use, florescein labelled siRNA and control transfection efficieny percantage starting after 2 hours of transfection till 24 hours is completed. after that, u can use PC and non-targeting siRNA before proceeding to your target gene. if you cant achieve at least 75% suppression with PC siRNA, then it is not necessary to try to silence your target gene..

just for an example, pc siRNA from Dharmacon targeting GAPDH with HyperFect, was silenced by 99% in my k-562 cells..hope these helps..

good luck..

I also prefer electroporation for suspension cells. If your transfection rate is very low in adherent cell, you also can try electroporation. In my experiment, I used Lipofectamine 2000 for adherent cell. Anyway, you can try commercial transfection reagent first. If it doesn't work well, you can come to electroporation.

try nucleofector machine. It works for difficult to transfect cells in suspension.

Thank you all for information. Once again I would explain that I have siRNA sequences which are working perfectly in adherent cells that's why I want to transfect the same siRNA in suspension cells. I am going to try transfection reagents first and if they did not work then I may switch to lentiviral transduction, or nucleofection. Thank you again......

hello, according to my lab. nucleofection is still the best among all the transfection method in suspension cells. the cell line we used in our lab is K562. normally can reach >60% transfection efficiency if using nucleofection.

Try to use lipofectamine 2000.

Best

In general siRNAs are more easy to get into cells than plasmids (eg. your GFP control). This of course does not necessarily mean that they will work efficiently as this will depend on where the siRNAs end up (ie if they are trapped somewhere in cells where they do not see their "target" complex).

I agree with above comments that nucleofection is BY FAR the most efficient way to transfect plasmids into cells. However, since the limiting step with plasmids often tends to be their nuclear transport while siRNAs generally need to be in the cytoplasm to be active this might not directly translate to making nucleofection (components of which apparently actively drive nuclear transport of the transfected complexes) the most efficient means to introduce siRNA. If you decide to go for plasmids (shRNAs) then use nucleofection (or viral vectors).

Hi,

as others mentioned, I think the best way to transfect cells in suspension is to use nucleofection. I've used loads of different cell lines using the appropriate Amaxa kit (Lonza) and the efficiency has always been quite high. In the past, I've been using the Lipofectamine 2000 (Lifetechnology) for Jurkat cells, for both overexpression of GFP-tagged proteins and siRNA-mediated silencing (1-10nM siRNA final concentration). You can find a protocol in here

(http://tools.lifetechnologies.com/Content/SFS/ProductNotes/F_Lipofectamine%202000b-040923-RD-MKT-TL-HL050602.pdf).

I've to say that, while the silencing of my proteins of interest was reasonably good, the overexpression didn't work that nicely.

Hope this helps.

@ Swat Kim Kerk, Ricardo Sánchez-Prieto , Aki Manninen and Elena Rainero .....I will try Lipofectamine 2000 and most probably Fugene HD first...and then may look the other way, if the knockdown efficiency is not sufficient enough. Thanx for your suggestions.

I would suggest electroporation or nucleofection...which is essentially the same.

Hi Imran,

Dreamfect has been sucessfully used for suspensino cells such as THP-1 cells,

for example: Wenzel M., J Immunol. 2012 Jan 1;188(1):394-403. Epub 2011 Dec 2.

Moreover, a new paper just came out about transfecting siRNA in THP-1 cells with DreamFect. "RNAi-mediated silencing of MLL-AF9 reveals

leukemia-associated downstream targets and processes" Fleischmann et al molecular Cancer 2014. here is the link

http://www.ncbi.nlm.nih.gov/pubmed/24517546

Let me know if you need information about this reagent [email protected]

I had beautiful knockdowns in suspension cells using Dharmacon's Accell siRNAs. there is a proprietary modification on the siRNA to help it get it. It works for sure every time but it is very expensive so small scale experiments are practical

We cannot say for sure it will work in all suspension cells, but ScreenFect A, which was recently developed by our research groups (Pavel Levkin's group and my own group) at KIT and is based based on new thiol lipid chemistry, is unique in functionally silencing genes in suspension cultures. See e.g. PMID: 23994267. Also siRNA transfection of very hard-to-transfect lymphoma cells apparently works well (80 % efficiency).

We commercialize it now (Incella.com), so contact us if you want a small sample to try.

[email protected].

Hi,

Hyperfect from qiagen is efficient for transfectibg suspension cells; especially k562. But since hl60 cells are very small, it is harder for them to reach and take into siRNA oligonucleotides. İ recommend you pipeting the transfected in every 3 hours in order to accelerate transfection efficiency. Transfection of Siglo risc free sirna or a positive control sirna (available in dharmacon) into your hl 60 cells will help you inorder to see transfection efficiency before proceeding with target gene.

Also, using modified sirna increase transfection efficiency ; ex: cholesterol conjugation

Good luck.

Hi,

I would really advice you to try Screenfect, as recommended by Dr Davidson above! We have tried it on diffuse large B-cell lymphoma cell lines with up to 85% transfection efficacy, and this without using harsh methods like electroporation. This transfection reagent is very mild to the cells, e.g. almost no disturbances on cell cycle phase distribution. However, there is great variation depending on which cell line you use, of course. I highly recommend you to read the excellent paper by Fischer et al 2013 describing Screenfect and as well as a nice flow cytometric method using Alexa Fluor®647 siRNA in combination with Calcein AM viability dye to screen for uptake of siRNA in cells (please see attached document, Materials and methods 3.2) Good luck! /T

Nucleofection appears to be superior in most cases of transient transfections that I've seen so far.

Imran,  I was wondering if you ever got your siRNA transfected into the Hl-60 cells?  and if so, what transfection method worked?

Jess Romanet....I tried lipofectamine and some other reagents but did not work....Luckily I got lentivirus from somewhere and that worked.......

hiperfect from qiagen is the best for either siRNA or miRNA transfection

I recommend Lullaby or Dreamfect, they have been successfully used to transfect siRNA into suspension cells, see examples of papers

http://www.ncbi.nlm.nih.gov/pubmed/17588944

http://www.ncbi.nlm.nih.gov/pubmed/24517546

http://www.ncbi.nlm.nih.gov/pubmed/22140256

If you want samples let me know at [email protected]

Best regards

For gene knockouts, presumably with siRNA, in HL-60 electroporation is one of the best routes to go (https://altogen.com/product/hl-60-electroporation-kit-plasmacytoma-cells-ccl240/). Although transfection efficiency will only be around 60%, you'll still have the necessary population of cells to conduct follow-up research on the effects of gene silencing.

Dear Muhammad,

In my opinion, you can take lentivirus-mediated shRNA knock-down into your consideration. Since it is difficult for suspension cells to be transfected using transfection reagents. Lentiviruses mediate integration of exogenous genes into host genome, and stable and long-term expression of exogenous genes, which may meet your demands.

Here is our transfection protocol with lentivirus in 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 200g 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.

Genemedi got a rich experience in virus production and transfection, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.