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.

Glial polyp of cervix

33F, a pedunculated polyp noticed in cervix during delivery of baby, 2 x 2 cm; strangely resembling cererbral cortical tissue. Rare, and nobody knows how. One of the most fascinating examples of a hamartoma.

Planets

Mars

Mars from Northern Hemisphere, 2115 hrs on 21st November 2020. Towards east, at Zenith. With a modest Telephoto lens of a Canon DSLR.

Uranus

As per astronomy charts for this time of the year (2230 hrs on Nov 21, 2020 in North India), this blue blob slightly east of zenith is supposed to be uranus.

Jupiter

As on 23 Nov 2020 at 1850 hrs from North India, Jupiter should be at south west 27 degrees above horizon.

Polymerase chain reaction and agarose gel electrophoresis of DNA

Electrophoresis run (blank) without PCR product, only control ('ladder') DNA.

Primer-dimers; note everything is less than 100 bp in size

Monitoring the progress of electrophoresis with bromophenol blue, which migrates at the same rate as a 150-300 bp DNA fragment

Seen on an UV transilluminator (blue band is bromophenol)

Often, one is not sure of the optimal temperature of a primer to anneal; in which case, trial and error at several temperatures is done. Modern thermocyclers allow different temperature for each row of wells (gradient PCR). The temperature with most product formation (thickest band) is selected.

Quantitative PCR for gene expression

Testing for gene expression is tricky; one needs to extract mRNA, reverse-transcibe it to cDNA and then run quantitaive (Q) PCR to determine copy numbers, with respect to some housekeeping gene (which is assumed to be equally expressed in healthy as well as diseased cells).

Here's interferon gamma cDNA expression from a healthy lymphocyte; note that the cycle threshold (Ct) is 28th cycle.

However, in a sick subject, IFNG is overexpressed, and determined from 22nd cycle onwards.

Determining difference in expression

To put a number on the mRNA expression between the two samples. one also needs to run a housekeeping gene like glyceraldehyde-3-phosphate dehydrogenase GAPDH. This would produce a table like the following:

Sample	Ct_IFNG	Ct_GAPDH	Difference in Ct
Healthy	28	22	6
Sick	23	21	2
Difference of differences in Ct			6-2 = 4

Thus, the sick sample has 2⁴ = 16 times more IFNG mRNA expression than healthy. (Remember that PCR cycles produce 2ⁿ copies after n cycles).

Is the product pure?

This particular run was carried with SYBR green, a dye which preferentially binds to double stranded DNA. Once we start heating the PCR products, at a certain temparature, the products denature, and SYBR green falls off, causing a drop of fluorescence. The sudden change in fluorescence is plotted in the melting curve.

Note that the y-axis is (negative) derivative of fluorescence, i.e. rate of change; thus the sudden drop is reflected in the peak

The melting curve from both samples show a single peak (pure product) which occur at the same temparature (indicating the same product in both runs).

Thanks: Dr Michael John,Sanskriti Rai, Pankaj Kumar & Uddep Chowdhury.