Titrating antibodies for flow cytometry

To determine the optimal dose of an antibody (which will bind cells at just the right proportion to generate a signal which is measurable within the limits of the lasers of the machine) is an exercise in perseverence. One must prepare serial dilutions of the antibody, isolate cells from blood (in this case, lymphocytes) and go on staining with each dilution of antibodies. 

Here, anti human FoxP3 has been prepared at doses of 5, 2.5, 1.25 and 0.625 microL (to stain one million cells, in each case). FoxP3 is a transcription factor expressed by a select set of T lymphocyte only upon stimulation by some external antigen. The graphs show staining intensity of the fluorochrome (in this case, phycoerythrin) as a histogram of cell counts, indicating expressionof FoxP3 inside the cells. Shift in median staining intensity has started to appear from 2.5 microL onwards. Note the difference between

• peaks of unstained cells (blue) and unstimulated cells (green), as expected
• peaks of stained cells, with increasing concentrations of antibodies, shown in progressively redder colors

The axis is logarithmic, so the differences between peaks are really much more than they seem

... as evident from the mean and median statistics (see legend of plot)

IL-17, the cytokine of acute inflammation, shows only a moderate increasewith stimulation.

Without stimulation, without staining for any antibody

Without stimulation, but with staining for any background IL-17 present

With stimulation; note the scattered points towards far right (high IL-17 expression)

With a constitutive surface marker like CD4, there is hardly any difference between unstimulated and stimulated

Orange - unstimulated, stained for CD4, green - stimulated and stained, purple- neither stimulated nor stained for CD4

Thanks Uddeep and Dr Abhinav Saurabh

Antibody screening for transplant

Screening for preformed antibodies

In solid organ transplant, preventing hyperacute rejection is the primary aim. The recipient serum is tested for antibodies against the commonest HLA antigens in the population ('panel'); however, the sheer number of different HLA antigens (about 15000 and ever increasing) make it a difficult test to be done manually.

In the solid phase bead assay, single/ multiple such antigens are attached to plastic beads and read by a specialised flow cytometer.

No anti-MHC antibodies in recipient serum; transplant can proceed

Non specific antibodies binding to beads, mostly against DQ and DP antibodies; transplant can proceed

High titre of antibodies against MHC Class II (DQ); contraindication to transplantatinon

Very high titre of antibdies against MHC Class I; a definite contraindication to transpant

Visualising cytokines

Cytokine production by cytotoxic T cells

T cells stimulated with tubercular antigen show a visible increase in the proportion of interferon-gamma producing CD8+ T cells; (flow cytometric data, PE Cy5 = CD8, FITC = IFN gamma)

Before stimulation; note the very small fraction of cells above the horizontal line (i.e. those producing interferon)

After stimulation; 2-4 % of cells have now been activated and moved above the line

The CD8 negative cell population (i.e. left of the vertical line) are a mix of B cells and helper (CD4+) T cells; note that CD4+ T cells will also produce interferons, as seen in the top left quadrant.