Understanding Fc blocking
Intact antibodies will bind to any cell that has an unoccupied Fc receptor. Only thymocytes and erythrocytes are void of Fc receptors on their cell surface. It is important to use an Fc block to bind up the Fc sites prior to incubation with antibodies. Cd16/32 specifically binds to Fc receptors and can actually be used in conjunction with a second ab to identify Fc receptor carrying cells. An example of using an Fc blocker is rat anti-mouse IgG2. Whole serum can be used but is less effective especially in cell lines derived from the hematopoetic system. You can select F(ab)'2 fragments of antibodies but these give weaker signals and may not all be commercially available. Unblocked samples will contain high levels of non-specific fluorescence and can often mask the true positive signal especially if that signal is weak.
Understanding dead cells
Dead cells have the ability to pick up free-floating fluorescence dyes. By using a dead cell marker such as Propidium Iodide or 7-AAD, cells that have a permeabilized nuclear membrane can absorb these dyes, which bind to nucleic acids. These dyes fluoresce brightly at 488-excitation and subsequently, can be excluded from data acquisition via live cell gating.
There are two kinds of binding: specific and non-specific. Specific activity includes Fab to epitope and Fc to Fc Receptor. Non-specific is an across-the-board low-affinity stain that is non-saturable. Antibodies don’t bind due to over-conjugation, non-purification, degradation of binding site or aggregation.
Understanding antibody dilutions
Most antibodies purchased from companies arrive with suggested working dilutions. Perform a titration on these antibodies to prove they work and see if you can get better resolution between positives and negatives by increasing the dilution factor. Start with the dilution the manufacturer recommends and the proceed with a twofold decrease (i.e. start with 1:100 then look at 1:200, 1:400, 1:800, 1:1600, etc.)
Understanding isotype controls
The appropriate negative control for an experiment is cells from each tissue labeled with a fluoresceinated anti-Ig from the same subclass of the monoclonal antibody. For example, mouse IgG1 or IgG2, flourochrome conjugated as needed. You must know the protein concentration of the isotype and specific antisera. If they are the same, then you can use the same dilution.
Always keep your cells in a buffer that contains some protein (antibodies like to cling to things and protein-free media causes the antibodies to cling to the tubes). Your medium should be free of calcium and magnesium to prevent clumping. THIS IS VERY IMPORTANT FOR CELL SORTING. It should also contain 0.5% serum or 1% BSA to keep them happy. Sodium azide is used to keep all media, which is being used at room temperature, bug-free. Cells that are returning to culture should not see any azide.
Procedure - direct staining single or multiple fluorophores
- In advance, prepare staining media (SM) made up of HBSS + 2% FCS.
- Harvest cells.
- Adjust concentration to 1-10 x 106.
- Wash once in HBSS and resuspend in 50 ul of appropriately diluted primary antibody cocktail (e.g. FITC cd4, pe cd8, cy5pe tcr).
- Incubate cells on ice for 30 minutes.
- Add 1 ml of SM.
- Insert glass Pasteur pipette into tube and add 300 ul whole serum into the pipette to provide a serum underlay.
- Spin cells at 1200 rpm for five minutes.
- Decant supernatant and resuspend cells in 500 ul of SM and 1 ul of 1 mg/ml stock Propidium Iodide (in water).
This method usually involves the addition of an unconjugated primary or a biotinylated primary. You will most likely need an FC blocker with this type of staining, depending on the tissue type. After the first spin as described above, resuspend the pellet of cells in 50 ul of the fluoresceinated second stage, appropriately diluted as per titration results or manufacturer's suggestion. Proceed with another 30-minute incubation on ice, followed by a serum underlay and centrifugation. FACS.