We developed polystyrene beads modified with the porphyrin TCPP as compensation controls in multicolor flow cytometry. The beads used in this protocol have fluorescence intensity comparable to TCPP stained cells and still pass through the flow cytometer. We accomplished that and are currently working on better methods to remove the particulate byproduct.
Porphyrin modified compensation beads were preferable to TCPP stain cells as compensation controls. These beads were stable over months without significant loss of fluorescence. Porphyrins are known to stain cancer cells selectively.
The research combines the selectivity of porphyrins with quantitative flow cytometry as a powerful platform for high throughput analysis of human samples. To begin, weigh 49.0 to 50.9 milligrams of TCPP using an analytical balance. Round the weight to one tenth of a milligram.
Set the measured amount of TCPP aside, protected from light. Based on the amount of TCPP, add the required amounts of purified water and isopropanol to a 100 milliliter glass beaker and cover it with parafilm to protect it from evaporation. Next, weigh the required amount of sodium bicarbonate based on the amount of TCPP and add it to the 100 milliliter beaker containing purified water and isopropanol.
Cover the solution with parafilm to protect it from evaporation and place it on a stir plate for approximately 10 minutes. Once the contents are dissolved, ensure that the pH of the solution is between nine and 10. Next, carefully add the weighted TCPP, protect the solution from light, and stir it for approximately 30 minutes until dissolved.
Store the solution in a polypropylene container at room temperature, protected from light. Begin by adding 4.3 milliliters of 0.1 molar MES buffer solution to a 15 milliliter polypropylene tube. Then add 288 microliters of the vortex stamene functionalized polystyrene bead suspension to the 15 milliliter polypropylene tube containing MES buffer.
Vortex the MES bead solution for 15 seconds at maximum speed. Next, vortex the one milligram per milliliter TCPP solution for 60 seconds at maximum speed. Then add 1.2 milliliters of this solution to the MES bead suspension.
After vortexing the resultant suspension for 15 seconds, cover the tube with foil. To begin, add freshly prepared 4.5 milliliters of EDC working solution to a 15 milliliter polypropylene tube containing the amine functionalized polystyrene beads and TCPP in MES buffer. Place the tube in an inverting rotator at 35 RPM for 16 hours at room temperature, protected from light.
Then centrifuge it for 10 minutes. Aspirate the supernatant and resuspend the beads in 0.8 milliliters of Hank's balanced salt solution. Then using a one milliliter pipette, transfer the bead solution to an amber polypropylene vial and store it at four degrees Celsius.
The relationship between the particulate formation, the median fluorescence intensity, or MFI, and the bead TCPP/EDC ratio showed that with increasing ratios, the MFI increased until it formed a plateau. The particulates appeared only when EDC was used as a coupling reagent during the labeling procedure. Labeling the beads with TCPP alone resulted in much lower fluorescence intensity.
When TCPP labeled beads were tested after 300 days, no significant change in the MFI or in the particulate content was observed. The coupling of TCPP to the beads had a minimum effect on its fluorescence emission spectrum. However, its spectrum differed more from TCPP labeled A549 lung cancer cells.
In a multiflora four labeled sputum sample, the TCPP labeled beads worked equally well as the TCPP stained A549 cells. Begin the quality check of the beads labeled as meso-tetra(4-carboxyphenyl)porphine or TCPP beads in the flow cytometer by running and collecting 10, 000 events of the rainbow beads. Then perform a rinse with water and collect 10, 000 events of the TCPP negative beads.
After rinsing with water again, collect 10, 000 events of the TCPP positive beads. Then perform a one minute water rinse before appropriate cleaning and shut down the flow cytometer. A representative outcome of the TCPP bead labeling process determined by flow cytometry is shown here with a standardized profile of rainbow beads.
These beads serve as quality checks for standardizing the laser voltages to detect TCPP. The light scatter profile of amine functionalized polystyrene, TCPP labeled beads was observed. The light scatter profile of non functionalized beads was used as a negative bead control to calculate the compensation matrix.
The population indicated by the red rectangle in the TCPP labeled beads was absent in the unlabeled beads suspension, representing that the TCPP related particulates were not formed in the absence of TCPP and EDC. The fluorescence intensity of the labeled beads selected by the black gate was several logs higher than that of the young labeled beads. Begin the TCPP bead filtration by diluting 0.8 milliliters of the TCPP bead suspension with 3.20 milliliters of ice cold Hank's balanced salt solution or HBSS.
Vortex the diluted bead suspension at maximum speed for 15 seconds. Then remove the plunger from a disposable five milliliter syringe and fit the syringe with a five micrometer glass fiber tip filter, having a 13 millimeter diameter. Next, add four milliliters of HBSS to the syringe before adding 0.5 milliliters of the vortexed diluted bead suspension.
Using the plunger, filter the bead suspension through the syringe filter at approximately two drops per second. Then wash the beads by drawing five milliliters of fresh HBSS into the syringe through the filter and pushing the HBSS out into the waste container at approximately two drops per second. To remove the beads from the filter, draw another five milliliters of fresh HBSS into the syringe through the filter.
Then remove the filter carefully from the syringe. After ejecting the bead suspension from the syringe into a 50 milliliter conical centrifuge tube, place the fresh filter back on the syringe and repeat the five milliliter HBSS wash four times. Once done, discard the filter and syringe.
Centrifuge the filtered bead suspensions at room temperature for 10 minutes at 1000 G.Aspirate the supernatant from each 50 milliliter tube. Gently resuspend the beads and 0.5 milliliters of fresh HBSS. Transfer the combined beads with a P1000 micro pipette to a new amber colored glass vial and store at four degrees Celsius.
The filtration effect on a bead suspension comprising approximately 66%particulates is seen here. After filtration, the particulate content decreased to approximately 25%The adverse effects of filtration are the loss of beads and a slight loss in fluorescence intensity.
