The overall goal of this procedure is to evaluate the potency of immunomodulatory drugs using an automated cell counter by measuring T lymphocyte blastogenesis or blast transformation. This assay provides a rapid method for quantifying T lymphocyte activation using an automated cell counter that is equipped to measure cell diameters. The main advantage of this technique is that it allows the quick simple and direct measurement from single cells unlike common T lymphocyte proliferation assays.

Treating lymphocytes with immunomodulatory drugs will either increase or decrease the rate and extent of cellular blastogenesis. Using the cell counter assay the extent of this blastogenesis can be measured in approximately four minutes per sample. Within ten minutes of sacrifice, spray the abdomen of the mouse with 70 percent ethanol and use scissors to make an incision in the fur and skin on the left side of the animal.

Hold the skin apart and back to reveal the peritoneum. Then apply a four percent chlorhexidine to the incision site. Using a second set of scissors and forceps, make a two to three centimeter cut along the central abdomen to open the peritoneum.

Then, remove the visceral attachments and excess fat surrounding the spleen and transfer the tissue into a container of DPBS. Next, add ten milliliters of complete RPMI 1640 medium to a ten centimeter culture dish and crush the spleens between two sterilized frosted glass slides. Filter the tissue slurry through a sterile 40 micron nylon cell strainer into a new ten centimeter culture dish to remove the connective tissues and debris and transfer the single-cell suspension to a 50 milliliter conical tube for centrifugation.

Resuspend the pellet in 20 milliliters of red blood cell lysis buffer. After ten minutes at room temperature with gentle rocking, collect the cells with another centrifugation and resuspend the pellet in ten milliliters of fresh medium. Then centrifuge the cells again for resuspension in two milliliters of 37 degree Celsius complete medium.

To purify the T cells, first wash one to two nylon wool columns per spleen two times with five milliliters of complete RPMI and place the columns in a 37 degree Celsius and five percent carbon dioxide cell culture incubator for one hour. At the end of the incubation, load two milliliters of the isolated splenocyte suspension onto the top of each column and allow the cells to pass through the column until the liquid reaches the top of the wool. Next, add two milliliters of fresh 37 degree Celsius complete medium to the top of the columns and allow the medium to pass through the nylon wool until the liquid at the top of the column reaches the top of the wool.

Now cover the wool with three milliliters of 37 degree Celsius complete medium and return the column to the cell culture incubator to all the B cells, fibroblasts and accessory cells to adhere to the wool fibers. After an hour, in a new 50 milliliter conical tube, wash the column two times with five milliliters of fresh complete medium to elute the non-adherent T cells. Then collect the cells by centrifugation.

After washing the T cells one time with ten milliliters of complete medium, resuspend the pellet in two milliliters of fresh complete medium. Then count the cells and dilute the suspension to a 0.5 times ten to the six cells per milliliter concentration in fresh complete medium for seeding into a six or 24-well cell culture plate as experimentally appropriate. Within 24 hours of their isolation, activate the nylon wool column isolated T cells with the appropriate stimulus and the experimental drug of interest.

After 12 to 72 hours, use a one milliliter pipette to gently mix the activated treated cells to remove any clumps. To measure the cell diameters by automated cell counter, transfer one milliliter of the treated cells from each well into individual sample cups and place the sample cups in the sample carousel of the automated cell counter. Enter the sample ID and cell type information to log the samples and begin running the samples.

After mixing trypan blue with the cell suspension, the cells are passed over an imaging field until about 100 cell images are collected from each sample. The software draws a circle around each detected trypan blue stained and unstained cell to determine the cell diameter. When all of the cells have been measured, the data are exported to a spreadsheet that shows the results as the total and viable cell counts for every measured diameter.

The data can then be presented as histograms. After two days of stimulation with PMA and ionomycin, a significant shift in the median of the frequency distribution towards bigger cell diameters with the number of T cells with smaller diameters reduced accordingly is observed. At a fixed 250 nanomolar ionomycin concentration, the PMA effect is not significantly changed by elevating the concentration of the activation stimulus from two to 250 nanograms per milliliter.

Nor is there an appreciable difference in the T cell proliferation observed. Calcineurin inhibitor drugs partially suppress both T cell blastogenesis and proliferation. T cell treatment with immunosuppressive compounds that target the calcineurin nuclear factor of activated T cells inhibit the blastogenic response by nearly 72 percent.

Rapamycin and FTY720 exhibit moderate but statistically significant effects on blastogenesis. While TRAM34 apparently does not affect murine T cell proliferation even at 700 nanomolar concentrations. When rapamycin and cyclosporin A are used together, blastogenesis is completely inhibited.

Similar results are observed with murine T cells activated with anti-CD3 and anti-CD28 conjugated magnetic beads. This assay can be used to successfully quantify the effects of various immunomodulatory drugs, giving a better assessment of drug potency compared to typical proliferation assays which also include contributions from apoptotic and necrotic cells. With this method, up to 15 samples can be measured in and out allowing for the simultaneous and subsequent quantification of both the blastogenesis and proliferation rates.

Occasionally air bubbles can get into the flow cell making the measurement unusable, therefore all images should be examined for air bubbles before the data from each trial are accepted for analysis. One limitation of this procedure is that if there's too much debris in the sample, measurement may treat the debris as viable cells. With suitable modifications, this assay has the potential to be adapted for studying the cell size change in neutrophils and hepatocytes.

Summary

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118 ICR PBMC