Hi Akshat, intuitively I would say BSA can stick to the plate/plastic as well. I do adhesion assays not using T-cells but CD34 cells or mononuclear cells on fibronectin-coated plates. Before plating cells, we wash cells off the medium with PBS and use an assay buffer free from serum or BSA.

You can use BSA-only-coated plate as a control or as a test expt to see what its doing on its own. Did you coat the plates with ICAM yourself or you have pre-coated plates from supplier, in the case of latter the supplier should be able to provide some info too.

Please keep us posted with what you see.

When I used to do these assays, we blocked with heat-denatured BSA.  I believe it does a better job of blocking.  You could also try a fancier blocking buffer like the Casein one from Pierce.  I have used this one successfully for ELISPOTS where sticking is a big problem. 

Dr. Jarajapu, I think that's a great idea. I'll try blocked vs. unblocked conditions, as well as assay buffers with or without BSA. Hopefully, these should be revealing of what's going on. I coat the plates myself with ICAM-1-Fc. 

Dr. Cantor, that's a good plan, too! We actually have some of the "protein-free" blocking buffer that we use for ELISAs. I'll definitely try that out, too.

Thank you, gentlemen! 

Best,

A.

Another idea - find an irrelevant protein (e.g. denatured or cleaved ICAM, or a different protein) to coat the "blank" well. This would take up some of the "space" on the plastic that is attracting the excess BSA and screwing up your experiment. 

That's a great idea, Dr. Glickstein! I shall definitely try that.

I ran the assay with blocked vs unblocked conditions. Sure enough, blocking reduces background. In both conditions, however, the T-cells show their greatest adhesion to blank wells. I think Dr. Glickstein's idea will be my next assay. 

Thank you all so much!

As it turns out, adhesion to ICAM-1-Fc is more of a symptom of T-cell activation than anything else. Overly activated T-cells and T-cells that are non-active/proliferative are not very adherent compared to T-cells which are "moderately" activated (I am sorry, I am doing an awful job of explaining this). But, my T-cell cultures are currently stable: the cells look succulent and are proliferative but they aren't in the initial burst.The adhesion assay appears to be working, even though the data is somewhat noisy. But, I think gentler washes (or practice with gentle washing) will lower that degree of background. 

I am really grateful for everyone's advice on here! Thank you!

A.

Thanks for updating us with your experiences. Really appreciate that.

Gentle washing: I dont know how reproducible is the gentle-ness of your washes from plate to plate or expt to expt, I am afraid your mood will have huge influence on it.

Instead, just make an SOP - with very finer details in this case - n follow it as muc as you could n once you know that the process is going smooth, you let someone else follow the protocol n reproduce qualitatively what you have seen. Thats the ultimate of validating an expt!!!

Have fun!

The mood thing is true! As is the amount of caffeine in my system! :-) One of the ways in which I am trying to make the "gentleness" of the washes more uniform is by using wide-bore pipette tips to reduce the amount of shear force exerted by wash buffer in the wells.

A.

Yeah! I like that! been there, done that. I remember cutting the tips to make the flow less vigorous n stressful to cells in a different context.

Anyhow, i wasnt kidding when I specifically mentioned on a public forum, these little little things matter a lot for a successful experiment AND WE NEVER PUBLISH THESE FINER DETAILS, never. So we have to get into conversations like this to make things work.

This is very true! One of the things I have come to appreciate in the pursuit of this PhD is that it truly is the allegedly "minor" things that make all the difference! 

How Long did you coat the plate with ICAM-1-Fc? 

Hello Akshat,

I am trying to perform this adhesion assay with ICAM-1 in the lab. Could you provide us the details you used for coating the plates (like concentration, temperature and time for incubation...)?

Thank you in advance.

Marta Lopes and Asma Ejaz The last time I ran this assay was in, oh, 2015? :-) But here is what I did:

1) I switched to untreated glass-bottomed plates from iBidi, and I acid-washed them with acid-alcohol (HCl and EtOH, but the concentrations escape me now).

2) Then, I'd coat the wells (or the chamber slide, for imaging purposes) with poly-L-Lysine at 0.1mg per ml: this you can coat at RT for 5-15 mins, wash off with sterile water, and let dry at RT overnight.

3) The saturating conc of recombinant human ICAM-1 was 5 micrograms per mL in my hands. I'd dilute this in PBS-0.1% BSA, and coat the slides/plates at RT overnight or at 37 for 2 hrs.

4) Wash off the ICAM with PBS, and block with PBS-5% BSA for an hour at 37C.

5) 3-4 washes with water.

6) Your cells are now ready to be seeded. Once you plate your cells, I like giving them a quick low speed spin: 30 seconds at the most. Literally, I set the centrifuge at 400xg, pop the plates in, hit start, and hit stop the moment it gets to 300. The centrifugal force gently pushes the cells down onto the surface enabling them to make quick contact with the ICAM-1.

6.1) I prefer to plate the cells in phenol red-free medium with 5% FBS/human AB serum (depending on your cell type). My favourite medium to do this is FluoraBrite. Should you choose to image the cells, the fluorescence is much clearer and sharper in this medium.

7) The plate should now be placed at 37C for 90-120 mins, after which they should be subject to 4 gentle washes with a warm (37C) wash buffer...which is essentially the medium you plated the cells in. I also recommend using wide-bore pipette tips for this and dripping the wash buffer from the side of the plate rather than directly blasting the cells with wash buffer. Shear force can sometimes loosen adhered firmly adhered cells, and we only want to get rid of those who cannotb commit to the ICAM-1. :-)

8) You can either pipette out the wash buffer, or just flick it out of the plate. Literally, pick the plate up, flip it over and with a gentle flick of your wrist, dispose of the wash buffer.

9) As far as a read out, there are few ways: I preferred fluorescence, but I hated CFSE because of how many cells it killed off, and I was working with a pretty rare population from PBMC anyway. Cell permeant dyes like CellTracker Green or Violet are gentler on the cells, so I recommend those.

10) Once you've performed your washes, you can pop the plate in a plate reader capable of reading fluorescence, and the magnitude of fluorescence is directly proportional to how many cells stuck around after washing, and thus can be thought of as a function of adhesion.

Certainly, +ve and -ve controls are vital for these assays with the view of subtracting background. I'd also include an unwashed control, where the cells are allowed to adhere, and are not washed. When read on the plate reader, the values this condition give you can be thought of as 100% or max adhesion, and you can normalise your treatment conditions as %of max to get a cleaner/crisper graph to wow everyone at lab meeting. :-)

All the best! I am sorry this is so scattered, but it's been a while! Feel free to reach out if there're questions, and I'll do my best to solve them.

Thanks for helping us Akshat Sharma

We are going to try with the details that you gave us.

I wish you all the the best!