We would like to thank David Rodriguez for assistance with the figure preparation and Precision Pathology Services (San Antonio, TX) for the use of its Navios EX flow cytometer.

All procedures need to be done using appropriate personal protective equipment.
1. Preparation of the TCPP stock solution, 1.0 mg/ mL
NOTE: This can be prepared monthly.
<ol>
	<li>Using an analytical balance, spatula, and weighing paper, weigh 49.0-50.9 mg of TCPP. Round the weight to 1/10 of a milligram. Set the measured amount of TCPP aside protected from light. 
	NOTE: Use a static gun if the weight reading is unstable.</li>
	<li>Determine the required amounts of purified water and isopropanol (IPA) from Table 1 based on the amount of TCPP weighed in step 1.1. Add the purified water and isopropanol to a 100 mL glass beaker, and cover with parafilm to protect from evaporation.</li>
	<li>Determine the required amount of sodium bicarbonate from Table 1 based on the amount of TCPP weighed in step 1.1.</li>
	<li>Using the analytical balance, spatula, and weighing paper, weigh the required amount of sodium bicarbonate determined in step 1.3. Round the weight to 1/10 of a milligram. 
	NOTE: Use a static gun if the weight reading is unstable.</li>
	<li>Add the sodium bicarbonate to the&#160;100 mL beaker containing purified water and isopropanol. Cover the solution with parafilm to protect from evaporation.</li>
	<li>Place the solution from step 1.5 on a stir plate, and stir until dissolved (approx. 10 min)</li>
	<li>Measure the pH to ensure that the sodium bicarbonate-containing solution from step 1.6 has a pH between 9 and 10.</li>
	<li>Slowly add the TCPP weighed in step 1.1 to the solution from step 1.7, and continue stirring until dissolved (~30 min). Protect from light during this step.</li>
	<li>Store in a glass or polypropylene container at room temperature and protected from light.</li>
</ol>
2. Preparation of 2-(N -morpholino)-ethanesulfonic acid (MES) and hemisodium salt buffer solution, 0.1 M, pH 6.0-6.2 (&#34;MES buffer&#34;)
NOTE: This must be prepared on the day of use and kept at room temperature.
<ol>
	<li>Weigh out 2.50 g of MES hemisodium salt, and add this to a 150 mL plastic bottle.</li>
	<li>Add 121 mL of purified water, and dissolve by manual shaking until no solid is visible.</li>
	<li>Measure the pH of the MES buffer to ensure that it is between 6 and 6.2.</li>
	<li>Maintain at room temperature for use on the same day.</li>
</ol>
3. N-(3-Dimethlyaminopropyl)-N&#39;-ethylcarbodiimide (EDC) powder
<ol>
	<li>Take the EDC powder out of the freezer, and let sit at room temperature until use in step 5.</li>
</ol>
4. Combining amine-functionalized polystyrene beads with TCPP solution
<ol>
	<li>Add 4.3 mL of the 0.1 M MES buffer solution prepared in step 2 to a 15 mL polypropylene tube.</li>
	<li>Vortex the amine-functionalized polystyrene bead suspension (10 &#181;m, 2.5% w/v) for 60 s at maximum speed.</li>
	<li>Add 288 &#181;L of this freshly vortexed bead suspension to the MES buffer from step 4.1.</li>
	<li>Vortex the MES/bead solution for 15 s at maximum speed.</li>
	<li>Vortex the 1 mg/mL TCPP solution prepared in step 1 for 60 s at maximum speed.</li>
	<li>Add 1.20 mL of this freshly vortexed TCPP stock solution to the MES/bead suspension from step 4.4.</li>
	<li>Vortex the MES/bead/TCPP suspension for 15 s at maximum speed.</li>
	<li>Cover the tube with foil while the EDC solution is prepared.</li>
</ol>
5. Preparation of N-(3-dimethlyaminopropyl)-N&#39;-ethylcarbodiimide (EDC) hydrocholoride (HCl) stock solution
NOTE: The EDC solution is perishable and should be used immediately following preparation.
<ol>
	<li>Add 20.0 mL of purified water to a new 50 mL conical tube.</li>
	<li>Weigh out 200 mg of EDC HCl from step 3, and add it to the water (step 5.1).</li>
	<li>Vortex the EDC HCL for 15 s at maximum speed to generate a clear solution.</li>
</ol>
6. Preparation of the EDC HCl/MES working solution
NOTE: The EDC HCl/MES solution is perishable and should be used immediately following preparation.
<ol>
	<li>Add 54.0 mL of MES buffer solution (prepared in step 2) to a 150 mL plastic bottle.</li>
	<li>Add 6.0 mL of the EDC HCl stock solution (prepared in step 5) to the MES buffer solution, and mix by shaking for 10 s.</li>
</ol>
7. Labeling the beads with TCPP
<ol>
	<li>Add 4.5 mL of EDC working solution (from step 6) to the 15 mL polypropylene tube containing the beads and TCPP in MES buffer (step 4.7).</li>
	<li>Place the tube in an inverting rotator at 35 rpm for 16 h at room temperature and protected from light.</li>
	<li>Centrifuge the tube at room temperature for 10 min at 1,000 &#215; g.</li>
	<li>Aspirate the supernatant, and resuspend the beads in 0.8 mL of Hanks&#39; balanced salt solution (HBSS).</li>
	<li>Transfer the bead solution to an amber polypropylene vial with a 1 mL pipette, and store at 4 &#176;C until further use. 
	&#8203;NOTE: The beads are stable for at least 3 months at this point.</li>
</ol>
8. Quality check (QC) of the TCPP beads by flow cytometry
NOTE: The QC should be centered on whether the median fluorescence intensity (MFI) of the TCPP beads is sufficiently bright for their intended use and the amount of particulates generated by the procedure. See the representative results section for more details.
<ol>
	<li>Label one 5 mL polystyrene tube as &#34;TCPP negative beads.&#34; 
	NOTE: The negative beads are different from the amine-functionalized beads used for labeling. See the Table of Materials.</li>
	<li>Label another tube as &#34;TCPP positive beads.&#34;</li>
	<li>Label another tube as &#34;Rainbow beads.&#34;</li>
	<li>Aliquot 300 &#181;L of ice-cold HBSS into the tubes labeled with &#34;TCPP negative beads&#34; and &#34;TCPP positive beads.&#34;</li>
	<li>Add 500 &#181;L of ice-cold HBSS into the tube labeled as &#34;Rainbow beads.&#34;</li>
	<li>Vortex the non-functionalized polystyrene (unlabeled) bead suspension briefly at maximum speed (2-3 s), and add 10 &#181;L of it to the tube labeled &#34;TCPP negative beads.&#34;</li>
	<li>Vortex the TCPP-labeled bead suspension (finalized in step 7.5) briefly at maximum speed, and add 3 &#181;L of it to the &#34;TCPP positive beads&#34; tube.</li>
	<li>Vortex the Rainbow bead suspension briefly at maximum speed, and add two drops of it into the &#34;Rainbow beads&#34; tube.</li>
	<li>Keep all tubes on ice, covered, and protected from light.</li>
	<li>Initiate the appropriate daily startup procedures of the flow cytometer, and perform a QC to verify the optimal fluids and laser alignment. 
	NOTE: For this part of the protocol, it is assumed that the operator is trained in the use of the flow cytometer that is available, including the procedures of standardizing the light scatter and fluorescence intensity, as well as the basic principles of calculating the correct compensation matrix.</li>
	<li>Run the Rainbow beads and the TCPP beads without changing the voltage settings in between the different runs.
	<ol>
		<li>Run and collect 10,000 events of the Rainbow beads.</li>
		<li>Perform a rinse with water, and collect 10,000 events of the TCPP-negative beads.</li>
		<li>Perform a rinse with water, and collect 10,000 events of the TCPP-positive beads.</li>
		<li>Perform a 1 min water rinse. 
		NOTE: It is important to perform a rinse with water after running the TCPP beads. If the TCPP is not rinsed from the lines in the cytometer, there is the possibility that residual TCPP can label cells in the next tube to be acquired.</li>
		<li>Perform the appropriate cleaning and shutdown protocols specific to the manufacturer&#39;s instructions for the cytometer. 
		&#8203;NOTE: For representative results, see Figure 1.</li>
	</ol>
	</li>
</ol>
9. Bead filtration
NOTE: If the QC of the beads by flow cytometry (step 8) shows a high proportion of particulates (70% or higher), consider filtering the bead suspension using the protocol below (Figure 2).
<ol>
	<li>Add 3.20 mL of ice-cold HBSS to 0.8 mL of the TCPP bead suspension finalized in step 7.5 (creating a five-fold dilution).</li>
	<li>Vortex the diluted bead suspension at maximum speed for 15 s.</li>
	<li>Remove the plunger from a disposable 5 mL syringe.</li>
	<li>Fit the syringe with a glass fiber tip filter (5 &#181;m, 13 mm diameter).</li>
	<li>Add 4 mL of HBSS to the syringe.</li>
	<li>Add 0.5 mL of the vortexed diluted bead suspension (step 9.2).</li>
	<li>Use the plunger to filter the suspension through the syringe/filter setup at approximately 2 drops/s.</li>
	<li>Wash the beads by drawing 5 mL of fresh HBSS into the syringe through the filter at approximately 2 drops/s.</li>
	<li>Push the HBSS out again into the waste container at approximately 2 drops/s.</li>
	<li>To remove the beads from the filter, draw another 5 mL of fresh HBSS into the syringe through the filter.</li>
	<li>Carefully remove the filter from the syringe.</li>
	<li>Eject the bead suspension from the syringe into a 50 mL conical centrifuge tube.</li>
	<li>Place the filter back on the syringe, and repeat steps 9.10-9.12 four more times. Then&#160;discard the filter and syringe.</li>
	<li>Repeat steps 9.2-9.13 until all the beads from step 9.1 have been filtered. Use a fresh syringe and filter each time.</li>
	<li>Centrifuge the filtered bead suspensions for 10 min at 1,000 &#215; g at room temperature.</li>
	<li>Aspirate the supernatant of each 50 mL tube, and gently resuspend the beads, combining them in 0.5 mL of fresh HBSS.</li>
	<li>Transfer the beads with a p1,000 micropipette to a new amber, glass, or polypropylene vial, and store at 4 &#176;C.</li>
	<li>Repeat step 8 to determine if the proportion of TCPP-related particulates in the filtered bead suspension has decreased. For representative results, see Figure 3.</li>
</ol>

Porphyrin-Modified Beads in Flow Cytometry

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+photodynamic+therapy:+New+probes+for+cancer+imaging+and+treatment.">Next-generation photodynamic therapy: New probes for cancer imaging and treatment.</a> Biochemistry. 57 (2), 173-174 (2018).</li><li>El-Far, M., Pimstone, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+28+porphyrins+and+their+abilities+to+localize+in+mammary+mouse+carcinoma:+Uroporphyrin+I+superior+to+hematoporphyrin+derivative.">A comparative study of 28 porphyrins and their abilities to localize in mammary mouse carcinoma: Uroporphyrin I superior to hematoporphyrin derivative.</a> Progress in Clinical and Biological Research. 170, 661-672 (1984).</li></ol>

All authors are employees of bioAffinity Technologies.

Despite the many applications of porphyrins in cancer diagnosis and therapeutics2, there is limited literature on their potential use as a flow cytometric reagent for the identification of cancerous versus non-cancerous cell populations in primary human tissues24,25,26. Our research on the flow cytometric analysis of human sputum24,27 requires the...

Porphyrins have been of interest for many years in the biomedical field owing to their fluorescence and tumor-targeting properties1,2,3. Therapeutic applications such as photodynamic therapy (PDT) and sonodynamic therapy (SDT) entail the systemic administration of a porphyrin to a cancer patient, the accumulation of the drug in the tumor, and the localized exposure of the tumor to a laser light of a specific wavelength or ultrasound. The exposure to laser light or ultrasound leads to the generation of reactive oxygen species by the porphyrin and subsequent cell death4,5. In photodynamic diagnosis (PDD), porphyrin fluorescence is used to distinguish cancer cells from normal cells6. In this context, protoporphyrin IX, a natural fluorescent porphyrin that accumulates in tumors upon the systemic or local injection of its precursor, 5-aminolevulinic acid (5-ALA), is used to identify gastrointestinal stromal tumors, bladder cancer, and brain cancer7,8. More recently, 5-ALA treatment was explored as an approach to detect minimal residual disease in multiple myeloma9. Our laboratory has been using the tetraaryl porphyrin TCPP (5,10,15,20-tetrakis-(4-carboxyphenyl)-21,23H-porphine) for its ability to selectively stain lung cancer cells and cancer-associated cells in human sputum samples, which is a property that has been exploited in slide-based and flow cytometric diagnostic assays10.
Some porphyrins are bifunctional in that they can be used as therapeutic and diagnostic agents2,11. In biomedical research, such bifunctional porphyrins are used to evaluate how their ability to selectively target and kill cancer cells is a function of their structure as well as how it is affected by the presence of other compounds12,13,14,15,16. Both the cellular uptake of porphyrins and their cytotoxicity can be measured on a flow cytometric platform in a high-throughput manner. The absorption and emission spectra of fluorescent porphyrins are complex, but most flow cytometric platforms are equipped to correctly identify them. The absorption spectrum of fluorescent porphyrins is characterized by a strong absorption band in the 380-500 nm range, known as the Soret band. Two to four weaker absorption bands are generally observed in the 500-750 nm range (Q bands)17. A blue&#160;488 nm laser, present in most flow cytometers, or a violet laser (405 nm) can generate light of the appropriate wavelength to excite porphyrins. The emission spectra of porphyrins typically display peaks in the 600-800 nm range18, which results in very little spectral overlap with fluorescein isothiocyanate or phycoerythrin (PE) fluorophores but considerable overlap with other often-used fluorophores, such as allophycocyanin (APC), as well as tandem fluorophores, such as PE-Cy5 and others. Therefore, when using porphyrins in multi-color flow cytometry assays, single-fluorophore controls are essential to adequately correct the spillover of fluorescence in channels other than the one designated to measure the porphyrin&#39;s fluorescence.
Ideally, the single-fluorophore controls used to calculate the spillover matrix for a panel of fluorophores (also called &#34;compensation controls&#34;) should consist of the same cell type(s) as the sample. However, using the sample for this purpose is not optimal if there is very little sample to begin with or if the target population within the sample is very small&#160;(for example, if one wants to look at minimal residual disease or cancer cells at the early stages of the disease). A useful alternative to cells is beads coupled with the same fluorophore that is used to analyze the sample. Many such beads are commercially available; these beads are either prelabeled with the desired fluorophore (prelabeled fluorophore-specific beads)19,20, or a fluorescently labeled antibody can be attached to them (antibody capture beads)20,21. While commercial compensation beads are available for many fluorophores, such beads are unavailable for porphyrins, despite their increasing use in basic and clinical research.
In addition to sample preservation and appropriately sized positive versus negative populations, the other advantages of using beads as compensation controls are the ease of preparation, low background fluorescence, and excellent stability over time22. The potential disadvantage of using beads as a compensation control is that the emission spectrum of the fluorescent antibody captured on beads may differ from that of the same antibody used to label the cells. This may be of specific importance when using a spectral flow cytometer20. Therefore, the development of beads as a compensation control needs to be performed on the flow cytometer that will be used for the assay for which the beads are developed. Moreover, the development of the beads needs to include a comparison with cells labeled with the same fluorescent staining reagent.
Here, we describe the preparation of TCPP amine-functionalized polystyrene compensation beads, whose median fluorescence intensity in the detection channel was comparable to that of TCPP-labeled cells in sputum, and their use as compensation controls for flow cytometry. The autofluorescence of equivalent, non-functionalized beads was sufficiently low for their use as negative fluorescence compensation controls. In addition, these beads demonstrated stability in storage for nearly 1 year.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amber plastic vials, 2 mL, U- bottom, polypropylene</td>
			<td>Research Products International&#160;&#160;</td>
			<td>ZC1028-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine-funtionalized polystyrene divinylbenzene crosslinked (PS/DVB) beads, 10.6 &#956;m diameter, 2.5% w/v aqueous suspension, 3.82 x 107 beads/mL, 7.11 x 1011 amine groups/ bead</td>
			<td>Spherotech</td>
			<td>APX-100-10</td>
			<td>Diameter spec. 8.0-12.9 um, suspension 2.5% w/v 3.82 x 107 beads/mL, 7.11 x 1011 amine groups/ bead</td>
		</tr>
		<tr>
			<td>Conical tubes, 50 mL, Falcon</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>with appropriate rotor</td>
		</tr>
		<tr>
			<td>Disposable polystyrene bottle with cap, 150 mL</td>
			<td>Fisher Scientific</td>
			<td>09-761-140</td>
			<td></td>
		</tr>
		<tr>
			<td>EDC (N- (3- dimethylaminopropyl)- N&#39;- ethylcarbodiimide hydrochloride), &#8805;98%</td>
			<td>Sigma</td>
			<td>03450-1G</td>
			<td>CAS No:&#160; 25952-53-8</td>
		</tr>
		<tr>
			<td>FlowJo Single Cell Analysis Software (v10.6.1)</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips, 22 x 22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-540-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass fiber syringe filters (Finneran, 5 &#181;m, 13 mm diameter)</td>
			<td>Thomas Scientific</td>
			<td>1190M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides, 275 x 75 x 1 mm</td>
			<td>Fisher Scientific</td>
			<td>12-550-143</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Fisher Scientific</td>
			<td>14-175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol, ACS grade</td>
			<td>Fisher Scientific</td>
			<td>AC423830010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical pipette, 1 channel, 100-1000 uL with tips</td>
			<td>Eppendorf</td>
			<td>3123000918</td>
			<td></td>
		</tr>
		<tr>
			<td>MES (22- (N- mopholino)- N&#39;- ethanesulfonic acid, hemisodium salt</td>
			<td>Sigma</td>
			<td>M0164</td>
			<td>CAS No:&#160; 117961-21-4</td>
		</tr>
		<tr>
			<td>Navios EX flow cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX-40 microscope with DP73 camera and 40X objective with cellSens software</td>
			<td>Olympus</td>
			<td></td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Pasteur pipettes, glass, 5.75&#34;</td>
			<td>Fisher Scientific</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter (UB 10 Ultra Basic)</td>
			<td>Denver Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette controller (Drummond)</td>
			<td>Pipete.com</td>
			<td>DP101</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Syringe, 5 mL</td>
			<td>Fisher Scientific</td>
			<td>14955452</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Particles (non-functionalized), SPHERO,&#160; 2.5% w/v, 8.0-12.9 &#181;m</td>
			<td>Spherotech</td>
			<td>PP-100-10&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes, 15mL, conical</td>
			<td>Fisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene tubes, round bottom&#160;</td>
			<td>Fisher Scientific</td>
			<td>14-959-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Rainbow Beads (Spherotech URCP-50-2K)</td>
			<td>Fisher Scientific</td>
			<td>NC9207381</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes, disposable - 10 mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-574</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes, disposable - 25 mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-576</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td>CAS No:&#160; 144-55-8</td>
		</tr>
		<tr>
			<td>TCPP (meso-tetra(4-carboxyphenyl)porphine)&#160; Frontier Scientific&#160;</td>
			<td>Fisher Scientific</td>
			<td>50-393-68</td>
			<td>CAS No:&#160; 14609-54-2</td>
		</tr>
		<tr>
			<td>Tecan Spark Plate Reader (or similar)</td>
			<td>Tecan Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube revolver/rotator</td>
			<td>Thermo Fisher</td>
			<td>88881001</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
	</tbody>
</table>

porphyrin-modified beads for use as compensation controls in flow cytometry

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or more fluorescent reagents, each functionalized with a different fluorescent molecule (fluorophore) that binds to cells selectively based on their phenotypic characteristics, such as cell surface antigen expression. The intensity of fluorescence from each reagent bound to cells can be measured on the flow cytometer using channels that detect a specified range of wavelengths. When multiple fluorophores are used, the light from individual fluorophores often spills over into undesired detection channels, which requires a correction to the fluorescence intensity data in a process called compensation. 
Compensation control particles, typically polymer beads bound to a single fluorophore, are needed for each fluorophore used in a cell labeling experiment. Data from compensation particles from the flow cytometer are used to apply a correction to the fluorescence intensity measurements. This protocol describes the preparation and purification of polystyrene compensation beads covalently functionalized with the fluorescent reagent meso-tetra(4-carboxyphenyl) porphine (TCPP) and their application in flow cytometry compensation. In this work, amine-functionalized polystyrene beads were treated with TCPP and the amide coupling reagent EDC (N-(3-dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride) at pH 6 and at room temperature for 16 h with agitation. The TCPP beads were isolated by centrifugation and resuspended in a pH 7 buffer for storage. TCPP-related particulates were observed as a byproduct. The number of these particulates could be reduced using an optional filtration protocol. The resultant TCPP beads were successfully used on a flow cytometer for compensation in experiments with human sputum cells labeled with multiple fluorophores. The TCPP beads proved stable following storage in a refrigerator for 300 days.

Author Spotlight: Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry  

Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry

We developed polystyrene beads modified with the porphyrin TCPP as compensation controls and multicolor flow cytometry. The beads used in this protocol have fluorescence intensity comparable to TCPP stain cells and still passed 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 stained cells as compensation controls. These speeds 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.

This protocol for the TCPP labeling of beads is relatively fast and efficient. Figure 1 shows a representative outcome of the TCPP bead-labeling process as determined by flow cytometry. Figure 1A shows the standardized profile of Rainbow beads, as detected in the appropriate channel for detecting TCPP. These beads serve as a QC for the standardization of the laser voltages for the detection of TCPP by the flow cytometer. Figure 1B s...

Watch this Scientific Journal Video about Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry at JoVE.com

The protocol describes how porphyrin-based compensation beads for flow cytometry are prepared by the reaction of amine-functionalized polystyrene beads with the porphyrin TCPP and the amide coupling reagent EDC. A filtration procedure is used to reduce the particulate byproducts.

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or ...

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1.4K Views.  bioAffinity Technologies. We developed polystyrene beads modified with the porphyrin TCPP as compensation controls and multicolor flow cytometry. The beads used in this protocol have fluorescence intensity comparable to TCPP stain cells and still passed 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 stained cells as compensation controls. These speeds were stable over m...

Video: Author Spotlight: Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry  

Preparation of Meso-Tetra(4-Carboxyphenyl) Porphine (TCPP) Stock Solution

To begin, weigh 49.0 to 50.9 milligrams of TCPP using an analytical balance. Round the weight to 1/10 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.

Combining Amine-Functionalized Polystyrene Beads with TCPP Solution

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 vortexed amine 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.

Labeling Amine-Functionalized Polystyrene Beads with TCPP

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 re-suspend 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 multi-flora four labeled sputum sample, the TCPP-labeled beads worked equally well as the TCPP-stained A549 cells.

Quality Check (QC) of the TCPP Beads by Flow Cytometry

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 bead 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 unlabeled beads.

TCPP Bead Filtration

Begin the TCPP bead filtration by diluting 0.8 milliliters of the TCPP bead suspension with 3.20 milliliters of ice-cold Hanks'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 1, 000g. 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 micropipette 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.