Many antibodies sell at $300 or less for 100 ug. That seems cheap to me as, where I am, that is what it costs to support a technician for two days or less. I was also taking into account that this requires no special equipment and can literally be done in an afternoon. I assume that your time is worth something : ) You could scale down if cost of antibody is really an issue. Go by surface area. So, a 60 mm dish would require about a third of what a 100 mm dish requires (and 1/3 as many cells). In the end, you have to decide if you want to use a proven method, or scrimp on antibody and possibly fail, in which case you will have thrown away your money and your time. As I stated, trypsinizing is not necessary. There is NO problem taking the cells off the plate as described. Let me repeat also, since this point seems to have been overlooked... DO NOT use a tissue culture plate to do this. Use a plastic PETRI DISH intended for bacterial work. This will NOT work if you use a tissue culture plate. The surface treatment applied to tissue culture plates causes reduced antibody binding and increased non-specific cell adhesion (plastic bacterial Petri dishes are more like ELISA plates in their surface chemistry). Mammalian cells generally will NOT grow on bacterial Pertri dishes, so they MUST be transferred to tissue culture plates. Also... please read the paper.

PBS is often used in sandwich ELISA applications to coat the capture antibody to the plate surface.It may also works for your purpose to.for detaching cells you should first consider the type of interaction between the Ab and the target which is a sum of weak interactions including hydrogen bonds, van der Waals forces, and ionic and hydrophobic interactions.

PBS is commonly used to coat the Ab's to a surface. Peprotech ELISA kits use this technique (below you can find a direction with an example of these kits). The capture antibody is diluted in PBS and this solution is added to a 96-well plates. After incubating overnight the Ab's are attached to the surface.

http://www.peprotech.com/Lists/PTCoA 900-K05%20Human%20EGF%20EDK%201107005.pdf

I have read no protocols about how to separate captured cells from non-captured cells. However, I investigated the adhesion of monocytes (cells grown in suspension) to myofibroblasts of colon (cells grown attached to a surface). The monocytes were stained with calcein, co-cultured with the myofibroblasts and the adhesion was measured by fluorescense. To eliminate non-attached cells the wells were washed with PBS twice and the results were good. You can try to clean the surface with PBS to remove non-attached cells and try to measure using some colorimetric method. In attachment you can find the article were it is described the adhesion.

If you have any doubt, please let me know.

If you want to remove cells from a plate that has been coated with antibodies, it is going to be dificult since antibodies are resistant to proteases (like trypsin). You could try using papain, but you will have to be careful: Cells will be digested as well. Use different low concentrations of papain and using the microscope you can determine the best timepoint and concentration to remove your cells. For testing if the cells are really dettaching, pipete the solution gently up and down in the well and check on the microscope. To check the viability, resuspend 10µl of your cell suspension in 90µl of trypan blue solution. If the cells turn blue (deep blue!), they got pores and got damaged. If not, just transfer them to a new plate with A LOT OF FRESH MEDIA WITH SERUM (to try to neutralize the papain) and let them grow as always. If everything goes well, between 5 and 12 hours your cells will have a partial or full adhesion to your plate.

The binding of the antibody I have never done it in cell culture plates. Only with ELISA plates, with a Carbonate buffer pH 9,6 or 9,2 (depending of the antibodies' host).

Alternative to papain: pepsinogen.

Don't try proteinase K. It is Too harsh and trypsin and thrombin do not work well on antibodies (at least not that I know of).

Good luck

You can use 0.1 M sodium bicarbonate buffer prepared in distilled water and PH-ed at PH 9.6. Filter solution to make it sterile (if needed). Coat the plate with Abs dissolved in 100ul of this solution per single well (96 well plate), leave for 2-4 hours at 37C in TC. If the plate will not be used immediately seal it tightly and preserve at 4C until further use. Wash the plate with the PBS (or media) 3x (this is to wash out unbound antibodies) and proceed with plating of your cells.

Detachment of the cells requires either trypsinization (use of 0.25% trypsin at RT) and incubation for 5 minutes at 37C or scraping of the cells by using cell scraper.

If you are working with Antibodies, use Carbonate Buffer (pH 9.6) and if you are working with Cells, use Phosphate Buffer (pH 7.2). Antibodies have to be coated on an ELISA Plate overnight (at least for 16-18 hours). Cells can be coated on a plate, by diluting them in PBS to required concentration and then Centrifuging the plate for about 7-10 mins at 1200-1500 rpm. Cells can be removed easily, using a mild Trypsin wash. Make sure you neutralize the trypsin solution immediatley which contains the cells, or centrifuge cells, discard trypsin solution and resuspend in fresh PBS.

Another trick to attach antibodies is to coat the surface of the wells first with Protein A from Staphylococcus aureus. Protein A binds the Fc portion of the antibody. Some commercial producers of ELISA plates use it; others do not. One just has to try it for one's own system.

Beware of one problem with mAbs from long-standing hybridoma lines. The mAb may go heavy-chain only and non-binding. I know this from experience. There are two sublasses if IgG in camelids that are heavy-chain only and functional, but they start that way.

One other thing to remember: Optimal coating of the plate with Ab is not necessarily optimal for binding the Ag or cells in question, because of steric hindrance.

I have performed a simlar assay, based an in house ELISA protocol.

I coated polystyrene sterile culture plates with antigen diluted in bicarbonate buffer (pH9.5) overnight. In the morning I washed the plates with PBS, blocked them then applied patient plasma to the plate-just like an ELISA, except I never used detergent and worked in a sterile environment. When incubation was complete I again washed the plates with PBS so that only antibodies specific to the antigen remained. I cultured with PBMC, and to remove cells vigourously washed with ice-cold PBS, then stained for flow cytometric analysis

At the first thanks a lot for your kindly respond

actually I wanna coat my plate(cell culture plate) with an specific Ab for isolation the specific cell

my goal is isolation the cells and after that I wanna detach the captured cells and expand them in culture medium

is it possible to culture the captured cells in this system or not?

Yes this is definitely possible. Just follow the same procedure which I mentioned for coating the Antibody on the plate. Incubate the Antibody plate overnight. Wash the plate thoroughly using a suitable wash buffer the next day (which should not interfere with your cell binding). Incubate your cell solution again overnight (possibly in a CO2 Incubator). This should capture your cells of interest and bind them to your antibody. Removing these bound cells will be a bit tricky...you can try the same trypsinization procedure, but you will need to check purity of cells (Preferably using a Flow Cytometer) and Viability and Cell Count. If you do have access to a Flow Cytometer Cell Sorter...it would possibly be a much easier method than an ELISA.

Coat 100 ug antibody on a standard polystyrene PETRI DISH (not a TC plate) 40 min. at room temp. in 50 mM Tris-HCl, pH 9.5. Rinse thoroughly, final rinse with 5% FBS in PBS (NOTE: all PBS is Ca/Mg free). Aspirate all liquid and add your cell suspension (washed in cold 5% FBS in PBS at 4 deg. (2 x 10^7 cells in 3 ml) and shake gently for 90 min. at 4 deg. Rinse gently (5x) with 5% FBS in PBS to remove non-captured cells. Pipette vigorously to collect captured cells (confirm in microscope). Cheap and easy. This method has been used for over 30 years. The generic term for this is "panning"... try googling "cell panning with antibodies" (you will get a lot of "inverse" panning hits, i.e. panning for antibodies with cells, but be patient and have a good look).

For example, see http://bloodjournal.hematologylibrary.org/content/65/1/190.full.pdf

Notes addressing some issues mentioned by others: Cell adhesion on the plastic dish itself is not an issue because you use Petri dishes, NOT tissue culture plates, you use Ca/Mg free PBS, you use a short incubation time (certainly not overnight (and you do everything at 4 deg. NOT 37 deg.). I have yet to see cells adhere to plastic under these conditions! Do a control plate with no antibody. You CAN trypsinize the cells if necessary. If you're not cutting the antibody, you're probably cutting the antigen. It does work. But vigorous pipetting does too (and so would scraping), since the cells are rounded and only loosely tethered by the antibodies (cells are huge compared to antibodies and the forces exerted by vigorous pipetting easily overcome the antibody attachments... maybe some patches of membrane are left behind, but the cells are generally in good health and recover nicely once they are restored to medium at 37 deg. If you need viable cells in the end, remember also to keep everything sterile.

To answer your most recent question, you cannot culture the cells in this system because the dish you are doing this in is a Petri dish. You need a surface-treated tissue culture dish, unless your cells are adhesion independent. It is easy to transfer the cells.

Good luck!

@Norbert,

I am sorry, but 100 µg of antibody called "cheap" can be done only if you have your own hybridoma cells and you produce your own antibody ;-)

Abot the Trypsin treatment, I don't agree. Many proteins are resistant to Trypsin. Like Integrins, EGFR, Ceacams, etc. Trypsin works because it damages the extracellular proteins and some other adhesion proteins, but if you have an specific antibody binding to a cellular protein, and this cellular protein is resistant to trypsin, there is going to be a problem to take them off the plate

@Fatemeh:

I could think, and I am just making a hypothesis, that if you would coat a cell culture plate with the antibody and allow the cells to bind at 4°C (in that Norbert is right: Binding at 4°C is much difficult, although after working with macrophages I think they might be able to challenge Norbert's idea of something binding to plastic at 4°C!) and then remove the not binding cells after a hour, and just leth them grow on the plate, the membrane turnover will remove eventually the proteins binding to the antibody from their membrane and they will be able to grow normally. Just a word of caution: It can happen that the binding of the antibody triggers the transmembrane pathways and signal cascades associated with it. Have always controls for your cell growth. Then you can evaluate any changes: good o bad.

Good luck

Many antibodies sell at $300 or less for 100 ug. That seems cheap to me as, where I am, that is what it costs to support a technician for two days or less. I was also taking into account that this requires no special equipment and can literally be done in an afternoon. I assume that your time is worth something : ) You could scale down if cost of antibody is really an issue. Go by surface area. So, a 60 mm dish would require about a third of what a 100 mm dish requires (and 1/3 as many cells). In the end, you have to decide if you want to use a proven method, or scrimp on antibody and possibly fail, in which case you will have thrown away your money and your time. As I stated, trypsinizing is not necessary. There is NO problem taking the cells off the plate as described. Let me repeat also, since this point seems to have been overlooked... DO NOT use a tissue culture plate to do this. Use a plastic PETRI DISH intended for bacterial work. This will NOT work if you use a tissue culture plate. The surface treatment applied to tissue culture plates causes reduced antibody binding and increased non-specific cell adhesion (plastic bacterial Petri dishes are more like ELISA plates in their surface chemistry). Mammalian cells generally will NOT grow on bacterial Pertri dishes, so they MUST be transferred to tissue culture plates. Also... please read the paper.

I think all these methods, i.e. Cell Panning with antibody, Dynabeads or Magnetic Cell Sorting, Cell sorting by FACS will work in their suitable situation. One can chose a method based on a particular application, material availability, and financial environment. I agree with Norbert's idea, it is relatively easy, lower in cost, etc.

Regarding Fatemeh's question, if a 96-well plate is preferable, I would suggest that, use a high binding ELISA plate with lid, coat the plate as you do in an ELISA experiment but handle it aseptically. After coating 4 - 16 hours, wash the plate with sterile PBS or TC medium, then add your crude cell solution (better with some FBS), incubate at 4C and follow Norbert's procedure. For a purer cell population, more round(s) of panning may be needed.