I agree with the comments from John, and would suggest that most of your cells are dead. You will not see it after the sorting but they do not like it (a lot of cavitation forces). If you have to redo the sorting tell the guys to use a larger nozle and decrease the pressure as much as possible, you will get less events per second but your will get more viable cells. When you sort try to harvest the cells in media with a high concentration of sera, do not add antibiotics during the harvesting (the cells can have real wholes and the internal concentration of some of the antibiotics will kill them). Regarding whether you can use the cells or not if they are in suspension it will depend a lot of what do you want to do with the cells (westernblot, prepare RNA, etc... if tey are alive is fine) if you want to do some functional assays you can be in trouble.

I hope it helps

Jose A.

I agree. First, wait to see if there are still some viable cells. Be patient - they may adhere eventually. Do not disturb them. If GFP expression is stable, you may grow the line back from very few cells. To improve survival, change the FACS settings as suggested. Also, make sure your cells are in exponential growth (less than 80% confluent), and dissociate them very carefully, before you start sorting. You've got more than 90% positives, maybe you don't need to sort at all...? Depends on your experminent of course. Good luck!

1) Look at your cells with a microscope if they look good

2) If you’re not sure stain them with To-Pro or Sytox (dead live staining)

3) If all is ok you can use poly-lysine, fibronectin or simply FBS coating over night before adding cells

4) HEK are quite strong and generally there is no problem for sorting this cells but pressure can be an option

5) The medium used for the sorting

6) Finally a biological problem with the GFP tagged protein (Use a control with HEK WT for the sorting)

Hi...

A few things to consider ||assuming the post-sort cells are well and truly alive||.

1) How did you dissociate the cells for your experiment? Some dissociation method can be too harsh such that it will affect cells attachment afterward. Furthermore, sorting is quite harsh on cells... Imagine being zap by a laser... you wont be too peachy after that.

Next, like what Jose said, the 2) nozzle size and 3) flow speed may be of concern.

In terms of 4) ECM, I guess it will help. A quick search of some company website showed that 2.5 ug/mL of Fibronectin may help (I hate using fibronectin presonally), that website also showed 5 ug/mL Collagen, as well as 10ug/mL Laminin, is comparable for HEK293 attachment.

I am going to assume that you are using a 5) serum supplemented medium, if not perhaps you can look at supplementing your culture medium with serum or to use freshly prepared culture medium...

Not sure if this will help, but I made use of a 6) HEPES buffered version of the medium I was using when I was doing FAC-sorting of some cultured cells.

In my experience (not 293 cells), I have FAC-sorted antibody labelled neural progenitor cells and got them growing again... but success only came after I tweaked the protocol --- countless time... Bottom line, it can be done, just need to use the O word... Optimize!!

Good Luck!!

Gary

Hello Priscilla,

I agree with my colleagues. Probably , like during primary culture establishment, if cells are weak, conditions to put them again in culture after cell sorting have to be non stressing. Also the cell viability verified and you have not to have too low concentration of cells/milliliter when subculturing them ...

I suggest you ook at the conditionsused by the following authors for primary culture of neural cells , after isolation by FACS. Perhaps it could be helpfull for you

Best regardes

Didier

Morrison, S.J., White, P.M., Zock, C., & Anderson, D.J. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell. 96, 737-749 (1999).

John is right. If the cells are alive and the medium contains FBS (10% or over), it might be caused by differentiation or phenotype drifting. Try to wash away DMSO when you thaw a new vial of cells and optimaze culture conditions.

Jinsheng

Yes, reduce the pressure, that did it for my cells...

Many of the above replies are right.....to my knowledge 293T cells were sensitive to over trypsinization or pressure.....so try handling them with little care.....you should find good results

My personnal experience with HEK293 T cells, led me to always coat the culture dish with gelatin. They seemed to have a hard time to adhere to the dish. So if your cells are healthy and alive after the sorting and you have been careful with all the conditions mentionned above, maybe simply try coating your dish. Good luck

I agree with the above replies. 293T cells is very sensitive to over trypsinization or pressure. To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal (ATCC). "

The type of kidney cell that the HEK293 cell line is unknown; and it is difficult to conclusively characterize the cells post-transformation, since adenovirus 5 could have significantly disrupted cell morphology and expression, and embryonic kidneys are a heterogenous mix of almost all the types of cells present in the body. In fact, it has been speculated by independent researchers, including Van der Eb himself, that the cells may be neuronal in origin. This is possible, although most cells derived from an embryonic kidney would be endothelial, epithelial, or fibroblasts. Neuronal origin is suspected because of the presence of the presence of mRNA and gene products typically found in neurons. HEK 293 cells have a very complex karyotype, exhibiting two or more copies of each chromosome and with a modal chromosome number of 64. They are described as hypotriploid, containing less than three times the number of chromosomes of a normal haploid human cell. Chromosomal abnormalities include a total of three copies of the X chromosome and four copies of chromosome N17 and N22."

All my cells died after being sorted (It was confirmed yesterday). I think the problem was so much pressure. I am not sure about the size of theses cells. But I think we need to increase the nozzle size and decrease the pressure. Other doubt is about the time that cells were kept in PBS and ice (about 4 hours). Could it be a problem? After sorting, I put the cells into DMEM medium plus FBS (10%) plus 0,2% pen/strep (antibiotic).

Approximately 15 µm diameter in suspension. Are you obliged to work at 4°C? Can you do it at RT? From my experiment they can stay alive in PBS during a night but at 37°C with HEPES (Or CO²) .

HEK cells are semi adherent in nature,so when HEK 293 cells are allowed to become overgrown, they loose some of their adherence. Then are also very sensitive to residual DMSO, so make sure you do a wash.Also grow the cells in lysine coated well plates. 293 cells are poorly attached. If you wish for tighter attachment you can also coat your plates with collagen.

do you mean you got 92% GFP +ve cells? did you do dead/live cell gating?

I agree with all the comments above: check the viability of your cells, the presence of serum, limit the pressure and handle with care. I will also add: check the age of your cells and do not split them too much at each passage. I would recommend 1:3 twice a week. Less (ie 1:10 once a week), they will lose their adherent phenotype very quickly. Also limit the number of passages and avoid overgrow, 2 ways to accelerate the passage of these cells in suspension (in fact each time they are stressed, they begin to float in the medium). If your are working from an ATCC vial, make one or two passages and freeze the cells at 90% confluency in vials of 2 to 5 million cells. After unfreezing (20% SVF, 1 vial per F25), we usually avoid to use them over P20. If your cells are already coming from an internal cell bank, then you will have to establish experimentally your maximum passage. I don't know what is your purpose, but you may know that 293T properties are different in suspension or in monolayers. For example, they become resistent to infection by some viruses

Once adherent cells if don't seem to attach to the flask indicates(with high probability) that the cell is dead. Avoid putting in ice for sure, HEK cells for me stay alive for about 2 hrs at max in PBS(0% FBS) AT ROOM TEMPERATURE, but putting in ice for 15 mins kills the cells(I have same experience last month).

It is a good discussion on 293 cells. I do only work with this cells for transfection because of its high transfection efficiency.

I have seen many people handling 293 cells as suspension culture, using serum free media to collect excretory proteins.

Agree. Maybe you had still trypsin in the suspension and did not antagonzie it with FBS.

Hi.

If viability is the issue, based on the protocol you described, I suggest to shorten the time needed to handle your cells. If you want to keep the 293 cold, perhaps consider putting it in the fridge than on ice (as ice is colder than your normal 2C-8C).

If you are only sorting for GPF, how come you need 4HRs? If you are doing other primary/secondary staining, I'd also suggest shorting the primary and secondary incubation time, in fridge and not on ice...

Next, for dissociation, I might try to use a milder version of trypsin. I like to use TryPLE Express, which is similar to trypsin but gentler on cells and do not need inactivation.

Also, do you have to do the live sort in PBS? What's stopping you from using culture medium (phenol-free version and perhaps with lower FBS to 1%-2%... or even 5%?) If you are worried the cells clump up because of the serum, use the 40um or 70um cell strainer before sorting...

Good Luck!!

I guess problem is with DNA delivery agent/solution. If dead/live cell gating was done while assessing? Trypsin must have been removed while sample preparation for FACS, or deactivated by serum after trypsinization. Also as mentioned by Gary Peh, sample should be prepared in PBS containing FBS (~2%).

We also had a similar problem when the GFP cells were sorted. The viability of the cells were poor and hardly any cells survived. The issue was overcome by increasing serum concentration of the medium in the collection tube when sorting. Also seeded at higher density.

So I would first replace the 70μm Nuzzle with 100 μm. Because 70μm Nuzzle is for Hek cells to small and is also Sample pressure is too high. Therefore, the sorted cells are probably not survived. So I will not use trypsin for Dissoziation of the cells but trypsin-free Dissoziation buffer and look after the sorting necessarily whether the cells are alive.

Check the following article on modulated HEK 293 adhesion/repulsion on a polymer called Parylene-C. I think you'll find it helpful:

http://onlinelibrary.wiley.com/doi/10.1002/jbm.a.34329/abstract;jsessionid=A298E8BBF38485F097A882135D4FF43A.d03t02

I worked more than 6 years with HEK293 cells, ya, it is surviving very less outside medium, detaches due to ph problem of the media and with minor shaking if culture is old..take care use fresh dishes