I want to deplete some subpopulations of immune cells from bone marrow with petri dish precoated with antibodies. But I don't know how to make it and how much antibody I need. Can anyone who has experience give me some advice, or the detailed protocol?
Hello Fei,
don't have experiences with that, but maybe Elispot is the right technique?
Look here:
http://www.aelvis.net/pdf/ELISPOTProtocolMice.pdf
or here: http://en.wikipedia.org/wiki/ELISPOT
Best,
Sven
It depends on the antibody itself. You can essentially perform an ELISA for cells. However, there should be something other marker based on which you could select the cells. You will be able to do such depeletions better with a MACS or a FACS system (Magnetism assisted cell sorting or Fluorescence assisted cell sorting)
MACS positive or negative separation column system would be the most ideal protocol. But if you have pre-coated Petri dishes then you should not worry about antibody concentration because it is already pre-coated. You would have to follow an ELISA protocol, in essence, WITHOUT azide and use 5-10% FCS in warm sterile PBS to keep the cells happy. Although, before all this, you need to check what cells you want to isolate in the end and check their adherence to the plastic of Petri dishes.
Oh... (silly me, I didn't read your question!) if you're coating a plate... try the concentration that's used in a MACS system and diluted your antibody in the FCS/PBS solution I mentioned earlier. Incubations - follow an ELISA protocol. Flush your cells by pipetting with the FCS/PBS solution.
I agree that the ideal approach to deplete subsets of immune cells from bone marrow is magnetic selection or FACS sorting. Nonetheless, if you have to coat petri dish, you should use your Ab diluted in sterile PBS (and check for concentration, I currently use 1ug/ml) WITHOUT serum, store at +4° ON, wash, saturate with PBS 1% FCS, wash and add your cell population.
I used to do 'panning' a lot - depleting CD8+ T cells from lymph node suspensions to get CD4+ T cells. The book that Peter L. mentioned Methods in Immunology is edited by Michell and Ishigi, if I recall correctly.
One correction to what Peter said - dilute the antibody in buffer with NO protein [it will bind to the petri dish and block the antibody binding]. Be sure to use petri dishes and not tissue culture plates.
The concentration of antibody you will need depends on whether it is a polyclonal or a monoclonal. If memory serves, we were using 1 microgram per milliliter of monoclonal anti-CD8 antibody, in a volume of 10 milliters.
The binding occurs via tyrosine residues in the antibody reacting with benzene rings in the polystyrene. This reaction occurs faster at alkaline pH. We diluted the anti-CD8 antibody in 50 mM Tris pH 9.5 for an hour at room temperature; alternately, if you want to use a neutral buffer like PBS, do it overnight at 4oC. If you use the high pH buffer, be sure to wash the plates multiple times with PBS to bring the pH back to neutral.
Cells with Fc receptors will bind to your antibody, so if such cells are present, you have to block the FcR before you pan the cells. For human cells, you can use an immune globulin product such as Gamma-gard. For mouse cells, you can use normal mouse serum
Hi,
usually petri dishes are made from polystyrene - the same material as microtiterplates used for ELISAs. That means, ELISA protocols would fit.
The easiest thing is to use a carbonate buffer with a pH between 10.5 and 11.5. Prepare 0.1 M stock solutions of NaHCO3 and NA2CO3 each and mix them until you reach the pH (careful - volumes could be 10:1). Block with complete medium afterwards (if it's serum free medium, use some protein solution for blocking).
It's right, use 0,1 mol/L Carbonate buffer pH 9.6, antibody concentration 1 - 10 µg/mL. Overnight, 4 °C. Blocking with Serum 1 - 3% in PBS, 1 - 3 h at room temp.
The antibody will bind via hydrophobic interaction to the polystyren from the dish and hydrophobic amino acids in the protein with a very high affinity.
using plates coated with the antibodies - this is the technology of the 80's.
Use either Stemcell technology ( tubes) or Miltelyi (columns) magnetic beads with antibody cocktails for selection. Use negative selection not to hurt the cells of interest. Purity should be between 95-98%.
http://www.miltenyibiotec.com/en.aspx
http://www.stemcell.com/
Of course panning with petri dishes is a bit old fashioned :-)
But you need no big investion and you can start at once. That's kind of advantage, too.
Or you can start to stuff you own columns with steel wool in syringes, like Miltenyi started it in the late 80s - you still need the magnetic beads then...
For most of antibodies, I use 10ug per 10cm dish (not dish for tissue culture, just regular petri dish) in 5 to 6 ml of PBS. Use Sodium Bicarbonate buffer (Ph 9-10) enhance binding of certain antibodies but not all. Leave it at 4C overnight and second day, wash them off with PBS or medium containing 5 to 10% FCS .
Great post. Is there a way to release captured cells? For example, i coat dish with F480 antibody to capture macrophages, incubate cell mixture on ice, rinse away unbound cells, then release antibody bound macrophages for subsequent RNA analysis. Let's presume there are no alternative methods for mac isolation (no beads, no flow, no adherence) please.
When I used to do this, we would release the bound cells with a scraper, or a low pH wash [3 ml of RPMI 1640 pH=3.0, ice cold, adjusted with acetic acid], collected into 12 ml D-PBS at pH 7.4. The brief exposure to low pH will dissociate the bound antibody from the cells and is not toxic, so long as you don't keep the cells in the low pH very long.
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