Dear Dr. Stavroula Zagkou

You may try the Ultra-Low Attachment microplate from Corning.

To reduce cell attachment as far as possible Ultra-Low Attachment surface coating has a very hydrophilic and neutrally charged hydrogel layer that is covalently linked directly to the polystyrene surface to prevent peeling. Since cell attachment proteins and other biomolecules passively adsorb to polystyrene surfaces through either hydrophobic or ionic interactions, this hydrogel surface naturally inhibits non-specific immobilization via these forces, thus inhibiting subsequent cell attachment. This surface is very stable, non-cytotoxic and biologically inert.

The details are provided in the link below.

https://ecatalog.corning.com/life-sciences/b2b/IN/en/Microplates/Assay-Microplates/96-Well-Microplates/Corning%C2%AE-96-well-Clear-Polystyrene-Microplates/p/7007

Regards,

Malcolm Nobre

Stavroula Zagkou Malcolm Nobre Genetic procedures expert here

I've dealt with this exact problem before.

If your cells are sticking to the 96-well plates like they're glued on there's a few things you might be able to do.

Get non-binding microplates.Greiner Bio-One and Thermo Fisher Scientific offer non-binding assay microplates that are specially designed to reduce surface adsorption. The plates are great because they have a hydrophobic surface, which prevents cells from sticking.

options like Greiner Bio-One's 96-well non-binding plates or Thermo Scientific Nunc™ non-binding plates. These plates are often made from materials like polystyrene and can come in different well shapes, like round-bottom or flat-bottom, depending on your preference and the type of assay.

Another one, which I'm impressed I was able to remember the name of. It was really going to bug me. I know it was a hydrogel coated plate. But anyway I'm talking about the "corning super low attachment plates" They are coated with a special hydrogel for the exact thing you were talking about.

Malcolm, I like your recommendation for pipette but I would buy a multichannel

Ependorf research pipette.

They are still stubborn, you could institute this in conjunction with Cell-Tak or other cell attachment media if you want to immobilize cells selectively. You combine the two, it might become very powerful method to help fix the problem you're having here. But sometimes you need to double up.

To prevent sticking non-binding options are what you really need to be focusing on.

If you didn't want to go the route of getting the plates with the hydrogel, you could try using plates coated with an anti-CD40 antibody can help if you want to focus on specific cell lines, what cell line are you working with? HEK? 293?

the Thermo Fisher Barnstead Lab-Line Shaker is a great choice of you set it to around 200 RPM during incubation to keep cells in suspension, using a multichannel pipette like the (Eppendorf Research plus) gentle washing

Try adding 2mM EDTA during washes to prevent cell clumping by chelating divalent cations that might promote cell adhesion. This can be a very dangerous to the structural integrity of the cells you are attempting to work with. So I would be careful, of any sticking like you described. If you are very careful it should be an easy to fix problem

To improve your washing protocol, if you are open to alteration of your procedure, using PBS with a slight pH adjustment, and perform multiple washes at room temperature to avoid cell shock.

The EDTA helps in detaching cells without damaging them. Keep the plates in a biosafety cabinet for an hour at room temperature post-seeding to stabilize the temperature across the plate, as thermal gradients can cause edge effects.

Perhaps it's just a volume problem, and you need a large volume platelike the Nunc 96 DeepWell plates are particularly useful if you're dealing with larger sample volumes or need to avoid cross-contamination.

Let me know if this information was helpful, and if it worked for you. I have a few other ideas, but it's kind of hard for me to remember the names of all the equipment. That was a fun exercise

Most certainly, if you use all these methods together you should see improvement.

Malcolm Nobre well I guess he has his answer. We should talk, I gave pretty much the same exact answer. I didn't even see your response, I had screenshotted this question in my phone earlier when I saw it in the feed. That's pretty funny, I'd like to talk to you you sound like you know what you're talking about.

Malcolm Nobre Alex Wolf Thank you both for your suggestions; I wil try the Ultra-Low Attachment microplates from Corning and the non-treated Nunc PS plates from Thermo.