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

William Bauta; Marcia Grayson; Rossella Titone; Jennifer Rebeles; Vivienne I. Rebel

doi:10.3791/65294

JoVE Journal > Chemistry

화학

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

Published: March 24, 2023

doi:

10.3791/65294

William Bauta¹, Marcia Grayson¹, Rossella Titone¹, Jennifer Rebeles¹, Vivienne I. Rebel¹

¹bioAffinity Technologies

Summary

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.

Abstract

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′-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.

Introduction

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 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's fluorescence.

Ideally, the single-fluorophore controls used to calculate the spillover matrix for a panel of fluorophores (also called "compensation controls") 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 (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.

Protocol

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.

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.
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.
Determine the required amount of sodium bicarbonate from Table 1 based on the amount of TCPP weighed in step 1.1.
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.
Add the sodium bicarbonate to the 100 mL beaker containing purified water and isopropanol. Cover the solution with parafilm to protect from evaporation.
Place the solution from step 1.5 on a stir plate, and stir until dissolved (approx. 10 min)
Measure the pH to ensure that the sodium bicarbonate-containing solution from step 1.6 has a pH between 9 and 10.
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.
Store in a glass or polypropylene container at room temperature and protected from light.

2. Preparation of 2-(N -morpholino)-ethanesulfonic acid (MES) and hemisodium salt buffer solution, 0.1 M, pH 6.0-6.2 ("MES buffer")

NOTE: This must be prepared on the day of use and kept at room temperature.

Weigh out 2.50 g of MES hemisodium salt, and add this to a 150 mL plastic bottle.
Add 121 mL of purified water, and dissolve by manual shaking until no solid is visible.
Measure the pH of the MES buffer to ensure that it is between 6 and 6.2.
Maintain at room temperature for use on the same day.

3. N-(3-Dimethlyaminopropyl)-N'-ethylcarbodiimide (EDC) powder

Take the EDC powder out of the freezer, and let sit at room temperature until use in step 5.

4. Combining amine-functionalized polystyrene beads with TCPP solution

Add 4.3 mL of the 0.1 M MES buffer solution prepared in step 2 to a 15 mL polypropylene tube.
Vortex the amine-functionalized polystyrene bead suspension (10 µm, 2.5% w/v) for 60 s at maximum speed.
Add 288 µL of this freshly vortexed bead suspension to the MES buffer from step 4.1.
Vortex the MES/bead solution for 15 s at maximum speed.
Vortex the 1 mg/mL TCPP solution prepared in step 1 for 60 s at maximum speed.
Add 1.20 mL of this freshly vortexed TCPP stock solution to the MES/bead suspension from step 4.4.
Vortex the MES/bead/TCPP suspension for 15 s at maximum speed.
Cover the tube with foil while the EDC solution is prepared.

5. Preparation of N-(3-dimethlyaminopropyl)-N'-ethylcarbodiimide (EDC) hydrocholoride (HCl) stock solution

NOTE: The EDC solution is perishable and should be used immediately following preparation.

Add 20.0 mL of purified water to a new 50 mL conical tube.
Weigh out 200 mg of EDC HCl from step 3, and add it to the water (step 5.1).
Vortex the EDC HCL for 15 s at maximum speed to generate a clear solution.

6. Preparation of the EDC HCl/MES working solution

NOTE: The EDC HCl/MES solution is perishable and should be used immediately following preparation.

Add 54.0 mL of MES buffer solution (prepared in step 2) to a 150 mL plastic bottle.
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.

7. Labeling the beads with TCPP

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).
Place the tube in an inverting rotator at 35 rpm for 16 h at room temperature and protected from light.
Centrifuge the tube at room temperature for 10 min at 1,000 × g.
Aspirate the supernatant, and resuspend the beads in 0.8 mL of Hanks' balanced salt solution (HBSS).
Transfer the bead solution to an amber polypropylene vial with a 1 mL pipette, and store at 4 °C until further use.
NOTE: The beads are stable for at least 3 months at this point.

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.

Label one 5 mL polystyrene tube as "TCPP negative beads."
NOTE: The negative beads are different from the amine-functionalized beads used for labeling. See the Table of Materials.
Label another tube as "TCPP positive beads."
Label another tube as "Rainbow beads."
Aliquot 300 µL of ice-cold HBSS into the tubes labeled with "TCPP negative beads" and "TCPP positive beads."
Add 500 µL of ice-cold HBSS into the tube labeled as "Rainbow beads."
Vortex the non-functionalized polystyrene (unlabeled) bead suspension briefly at maximum speed (2-3 s), and add 10 µL of it to the tube labeled "TCPP negative beads."
Vortex the TCPP-labeled bead suspension (finalized in step 7.5) briefly at maximum speed, and add 3 µL of it to the "TCPP positive beads" tube.
Vortex the Rainbow bead suspension briefly at maximum speed, and add two drops of it into the "Rainbow beads" tube.
Keep all tubes on ice, covered, and protected from light.
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.
Run the Rainbow beads and the TCPP beads without changing the voltage settings in between the different runs.
1. Run and collect 10,000 events of the Rainbow beads.
2. Perform a rinse with water, and collect 10,000 events of the TCPP-negative beads.
3. Perform a rinse with water, and collect 10,000 events of the TCPP-positive beads.
4. 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.
5. Perform the appropriate cleaning and shutdown protocols specific to the manufacturer's instructions for the cytometer.
  NOTE: For representative results, see Figure 1.

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).

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).
Vortex the diluted bead suspension at maximum speed for 15 s.
Remove the plunger from a disposable 5 mL syringe.
Fit the syringe with a glass fiber tip filter (5 µm, 13 mm diameter).
Add 4 mL of HBSS to the syringe.
Add 0.5 mL of the vortexed diluted bead suspension (step 9.2).
Use the plunger to filter the suspension through the syringe/filter setup at approximately 2 drops/s.
Wash the beads by drawing 5 mL of fresh HBSS into the syringe through the filter at approximately 2 drops/s.
Push the HBSS out again into the waste container at approximately 2 drops/s.
To remove the beads from the filter, draw another 5 mL of fresh HBSS into the syringe through the filter.
Carefully remove the filter from the syringe.
Eject the bead suspension from the syringe into a 50 mL conical centrifuge tube.
Place the filter back on the syringe, and repeat steps 9.10-9.12 four more times. Then discard the filter and syringe.
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.
Centrifuge the filtered bead suspensions for 10 min at 1,000 × g at room temperature.
Aspirate the supernatant of each 50 mL tube, and gently resuspend the beads, combining them in 0.5 mL of fresh HBSS.
Transfer the beads with a p1,000 micropipette to a new amber, glass, or polypropylene vial, and store at 4 °C.
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.

Representative Results

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 shows the light-scatter profile of amine-functionalized polystyrene, TCPP-labeled beads. Figure 1C shows the light-scatter profile of non-functionalized beads (unlabeled beads); we use these as a negative bead control to calculate the compensation matrix. The population indicated by the red rectangle, which is absent in the unlabeled bead suspension, represents the TCPP-related particulates formed when TCPP is exposed to the coupling reagent EDC. Such aggregates should be excluded when the fluorescence intensity (FI) of the beads is determined. Figure 1D shows the FI of the labeled beads, selected by the black gate in Figure 1B, which is clearly several logs higher than the FI of the (ungated) unlabeled beads (comparing Figure 1D 세스 Figure 1E).

The proportion of particulates (i.e., the events indicated by the red gate in Figure 1B) may vary per labeling batch. For most standard flow cytometry applications, a bead suspension with 50% particulates or less allows one to correctly set up compensation in a multi-color flow cytometry experiment using TCPP. There are applications though, for example, where automated data analysis is used, for which much higher particulate content may interfere with proper compensation settings. In such cases, filtration is recommended (protocol step 9 and Figure 2). Figure 3 shows the effect of filtration on the bead suspension that comprised ~65% particulates (left panels of Figure 3A,B). After filtration, the particulate content decreased to ~12%. The adverse effects of filtration are some loss of beads (not shown) and a slight loss of FI (Figure 3C). These effects should be taken into account when filtration of the bead suspension is being considered. An alternative to bead filtration to lower the particulate content is to alter the ratio between the beads, the TCPP, and the coupling reagent EDC. However, alterations in these ratios also affect the FI of the beads.

The relationship between the particulate formation, MFI, and bead/TCPP/EDC ratio is illustrated in Figure 4 and Figure 5. Figure 4A and Figure 5A show the MFI of the labeled beads as a function of the EDC/TCPP ratio and the TCPP/bead ratio, respectively. With increasing ratios, the MFI increases until it plateaus. The representative examples in Figure 4B and Figure 5B show that the particulate content increases with the respective increasing ratio. This TCPP labeling protocol was optimized for maximum MFI while keeping the particulate content as low as possible (arrows in Figure 4A and Figure 5A). The particulates only appear when EDC is used as a coupling reagent during the labeling procedure. Using only beads and TCPP, particulates are not formed (not shown), but the labeling of the beads results in beads with much lower FI (comparing Figure 6A with Figure 6B). We tried to replace this EDC-based coupling method with a method based on an activated NHS (N-hydroxysuccinimide) ester of TCPP²³. However, we found no significant difference in FI using this alternative coupling method compared to just using beads and TCPP together, suggesting there is negligible covalent binding of TCPP to the beads with this method (comparing Figure 6B with Figure 6C).

The data presented in all figures thus far were collected using 10.6 µm diameter beads. We hypothesized that larger beads may have more TCPP binding sites due to the increased surface area and could, therefore, display higher FI. Conversely, we hypothesized that smaller beads with smaller surface areas and fewer TCPP binding sites should display lower FI. Using the same labeling protocol as described here but with 8.6 µm beads or 20 µm beads (instead of 10.6 µm beads), we found the predicted reduction in FI in the 8.6 µm beads compared to the 10.6 µm beads (comparing Figure 6D with Figure 6A). The 20 µm beads displayed an FI off the scale for the flow cytometer we used (data not shown), but these larger beads settled out of solution quickly thereafter and were not pursued further.

This protocol for labeling beads with TCPP was developed for the purpose of having a reliable compensation tool in flow cytometry experiments where TCPP is used to label cells. In the past, we used A549 cells for this purpose²⁴, but the labeling procedure did not always result in a high FI. Furthermore, the cells, even after fixation, could not be used for more than 1 month. The protocol for labeling (10.6 µm) beads with TCPP described herein consistently resulted in brightly stained beads, as shown in Figure 1. Moreover, when the same TCPP-labeled beads were tested by flow cytometry after 300 days, we did not observe a significant change in the MFI nor in the particulate content (Figure 7). The coupling of TCPP to the beads had little effect on its fluorescence emission spectrum (Figure 8A; comparing the dashed black line to the solid black line, which represents the fluorescence emission spectrum of TCPP in solution). The fluorescence emission spectrum of TCPP-labeled beads differed more from the spectrum of TCPP measured inside A549 lung cancer cells, especially around the 700-725 nm wavelengths (Figure 8A; dashed black line compared to the orange line, respectively). However, with the ability to compensate the TCPP signal from the other signals in a multi-fluorophore-labeled sputum sample, the TCPP-labeled beads worked equally well as the TCPP-stained A549 cells (comparing Figure 8B with Figure 8C, respectively).

The .LMD files can be found in the FlowRepository (https://flowrepository.org), under the IDs FR-FCM-Z6ZP (Figure 1); FR-FCM-Z5UJ (Figure 3B,C); FR-FCM-Z6ZR (Figure 4B); FR-FCM-Z6ZS (Figure 5B); FR-FCM-Z6ZB (Figure 6); FR-FCM-Z6Y5 (Figure 8B); and FR-FCM-Z6ZT (Figure 8C).

Figure 1: Quality check of the beads labeled with TCPP. (A) Rainbow beads. Presented is the histogram of the Rainbow beads as detected in the channel specific for the APC fluorophore. This channel can also be used to detect TCPP fluorescence. The Rainbow beads serve as a QC for the APC laser; the first and fourth peaks should fall in the region indicated by the respective brackets. (B,D) TCPP-labeled beads. (C,E) Unlabeled beads. This particular sample of TCPP-labeled bead solution included 51.9% beads (black rectangle gate in B). The events in the red rectangle represent the particulates that formed during the TCPP labeling. Such particulates were absent in the unlabeled bead solution (comparing the red rectangles in B and C). The fluorescence intensities of the TCPP-labeled and unlabeled beads are presented in D and E, respectively. Abbreviations: SSC = side scatter; FSC = forward scatter; APC = allophycocyanin; TCPP = meso-tetra(4-carboxyphenyl) porphine. Please click here to view a larger version of this figure.

Figure 2: Schematic representation of the bead filtering method used in protocol steps 9.3-9.12. Please click here to view a larger version of this figure.

Figure 3: Effects of filtration on particulates and fluorescence intensity. (A) Microscopy images of 10 µm beads labeled with TCPP before (left picture) and after filtration (right image). The images were taken at a 20x amplification. Scale bar = 20 µm. (B) The TCPP-bead suspension prior to filtration (left profile) included 25.6% beads (black rectangle) and 65.7% debris or particulates (red rectangle). The proportion of beads in the suspension increased to 74.7% after filtration (right profile; black rectangle), and the debris decreased to 11.9%. (C) Histograms of the TCPP FI of the beads as identified in B. The histogram on the left represents the TCPP-labeled beads before filtration, with an FI slightly higher than that of the beads after filtration (histogram on the right). The red lines, placed at an FI of 1 × 10³ in both figures, are to facilitate the comparison of both histograms. Abbreviations: SSC = side scatter; FSC = forward scatter; APC = allophycocyanin; TCPP = meso-tetra(4-carboxyphenyl) porphine; FI = fluorescence intensity. Please click here to view a larger version of this figure.

Figure 4: Relationship between the EDC/TCPP ratio, median fluorescence intensity (MFI), and particulate formation. (A) Presented is the relationship between the EDC/TCPP ratio used in the bead labeling protocol and the MFI of the ensuing TCPP-labeled beads. The curve shows the average MFI of three independent experiments ± standard deviation (SD). The red arrow (3.75) indicates the volumetric ratio of the EDC and TCPP solutions used in this protocol. (B) Representative light scatter profiles of the bead suspensions prepared with the EDC/TCPP ratios as indicated above each profile. All the profiles are derived from the same experiment. The beads are indicated by the black rectangle. The particulate formation, indicated by the red rectangles, increases with increasing EDC/TCPP ratios. Please click here to view a larger version of this figure.

Figure 5. Relationship between the TCPP/beads ratio, MFI, and particulate formation. (A) Presented is the relationship between the TCPP/beads volumetric ratio used in the bead labeling protocol and the MFI of the ensuing TCPP-labeled beads. The MFI increases quickly, reaching a plateau phase after a TCPP/beads ratio of 2. The curve shows the average MFI of three independent experiments ± standard deviation (SD). The red arrow (4.17) indicates the ratio used in this protocol. (B) Representative light scatter profiles of the bead suspensions prepared with the TCCP/beads ratios as indicated above each profile. All the profiles are derived from the same experiment. The beads are indicated by the black rectangles. The particulate formation, indicated by the red rectangles, increases with increasing TCPP/bead ratios. Please click here to view a larger version of this figure.

Figure 6: A comparison of the coupling methods and bead sizes used. Shown are histograms of the fluorescence intensity of the beads labeled with TCPP. (A) The 10.6 µm beads were labeled according to the protocol provided, including the use of the coupling reagent EDC. (B) The same as (A), but EDC was omitted during the labeling protocol. (C) The 10.6 µm beads were labeled using the NHS ester, according to the method described by Kabe et al.²³. (D) Same as (A), but instead of 10.6 µm beads, 8.6 µm beads were used. The vertical red lines in each profile are arbitrarily set at 1 × 10³ to facilitate comparisons between the histograms. Please click here to view a larger version of this figure.

Figure 7: Stability of the TCPP beads over time. TCPP-labeled beads (10 µm) were labeled according to the protocol, stored in the refrigerator at 4 °C, and kept in the dark. Day 0 is the day of labeling. At various time points thereafter, aliquots of the stored beads were reanalyzed by flow cytometry. Presented are measurements of the (A) MFI and (B) percentage particulates relative to the day 0 value, which was arbitrarily set at 100%. The horizontal orange lines represent the values 10% above and 10% below those at day 0. Please click here to view a larger version of this figure.

Figure 8: TCPP beads compared to TCPP-stained cells as compensation tools. (A) The fluorescence emission spectra of TCCP in solution, TCPP coupled to beads, and TCPP after the staining of A549 lung cancer cells were measured on a microplate reader. The emission spectra of soluble and bead-bound TCPP are very similar. Both differ from that of labeled A549 cells around the 700 nm wavelength. (B,C). A sputum sample was processed into single cells and labeled with multiple fluorophores: AF488 (FL1), BV510 (FL10), PE (FL2), PE-CF594 (FL3), and TCPP (FL6). For each fluorophore, we prepared single-color control tubes containing beads coupled with the relevant fluorophore. For TCPP, two control tubes were set up: one with TCPP-labeled beads and one with fixed A549 cells labeled with TCPP. This allowed us to create two compensation matrices: (B) one in which the TCPP-labeled beads were used as the compensation control for TCPP (FL-6) and (C) one in which TCPP-labeled A549 cells were used. These two compensation matrices are very similar, with the largest difference in TCPP-related compensation between TCPP (FL6) and FL-3. The accompanying dot plots that display these particular parameters show near-identical profiles. Please click here to view a larger version of this figure.

Table 1: Reagents required for the TCPP stock solution. The quantities of NaHCO₃, purified water, and isopropanol (IPA) are based on the amount of TCPP weighed in step 1.1 (between 49.0 mg and 50.9 mg). Please click here to download this Table.

Discussion

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 staining of dissociated sputum cells with TCPP and several other fluorophores. However, fluorescence spillover of TCPP in the detection channels designated for other fluorophores meant that a TCPP-labeled bead was needed as a compensation control. However, no such bead was commercially available and had to be developed. The attachment of tetraaryl porphyrins to silicon dioxide-based molecular sieves²⁸^,²⁹ has been reported. However, silicon dioxide-based particles proved unsuitable for the purpose of preparing flow cytometry standards (data not shown). Therefore, our studies focused on polystyrene beads.

A solution of TCPP in isopropanol-water (1:1 v/v)³⁰ labeled the polystyrene beads at pH 6, but the MFI was too low relative to the stained cells in our assay. We explored covalent bead modification as a way of increasing the MFI. The covalent modification of the amine-functionalized latex beads with the NHS ester of TCPP through amidation was previously described by Kabe et al.²³ using non-commercial 2 µm diameter beads³¹. In the study by Kabe et al.²³ , TCPP was incubated in DMF with N-hydroxysuccinimide (NHS)³² for 5 h to prepare an activated NHS mono-ester of TCPP. This activated ester solution was then reacted with beads containing a terminal amine linker. However, when we applied this protocol to commercial amine-functionalized polystyrene beads, the labeling resulted in beads with an MFI insufficiently bright for compensation. Therefore, we explored the direct amidation of amine polystyrene beads without using an NHS ester intermediate.

In this work, amine-functionalized polystyrene beads were treated with TCPP in the presence of the amide coupling reagent EDC to generate amide bonds between the amino groups on the beads and the carboxy groups on the TCPP. EDC was selected because of its aqueous solubility, proven utility in aqueous amidation reactions over a wide pH range, low cost, and water-soluble by products³³^,³⁴. We reacted amine polystyrene beads with diameters of 8.6 µm, 10.6 µm, and 20 µm with TCPP and EDC in water at pH 6. Earlier studies have indicated that TCPP remains soluble at this pH, and this pH is also compatible with EDC³³. The labeled 8.6 µm beads resulted in an MFI that was too low for our assay. The 20 µm beads had a sufficiently high MFI but were prone to settling out of suspension and clogging the flow cytometer (not shown). The 10.6 µm beads had an adequate MFI, remained in suspension long enough for flow cytometry analysis and thus were optimized as compensation standards. Notably, the equivalent unlabeled beads had low background fluorescence and could be used as negative control beads for compensation.

Increasing the volumetric reagent ratio of EDC to TCPP while keeping the quantity of beads constant increased the bead MFI until a plateau was reached at a ratio of about 2. An MFI plateau was also observed as the ratio of TCPP to beads was increased while holding the EDC to TCPP ratio constant. Since the TCPP labeled the beads in the absence of EDC, the MFI plateau may be a result of covalent and non-covalent binding sites being saturated on the surface of the bead. A TCPP-related particulate byproduct was observed when EDC and TCPP were combined at pH 6. This particulate had a fluorescence spectrum similar to TCPP but did not form in the absence of EDC, suggesting that it is an insoluble byproduct from the reaction of EDC with TCPP. The particulates were irregularly shaped and, while generally smaller than 5 µm, could form larger aggregates and could also adhere to the surface of the beads. TCPP-related particulates were also observed when amine polystyrene beads were reacted with TCPP and EDC. The amount of particulate increased with both a higher EDC to TCPP ratio and a higher TCPP to beads ratio. The reagent ratios selected in the final protocol are key to the success of the bead labeling procedure because they ensure that the resulting bead suspension has a high MFI while the particulate level remains acceptable. The level of particulates produced in this protocol does not preclude the use of TCPP beads for compensation, but higher levels of particulates can hamper the accuracy of the compensation settings³⁵. We also developed a filtration procedure that can reduce the level of particulates significantly should these be present at a high level.

We conducted our reaction using a rotating agitator. Not agitating the reaction results in slower staining (data not shown). A 16 h reaction time is recommended. Longer reaction times (>24 h) result in higher levels of TCPP particulates, while shorter reaction times (<8 h) result in lower bead MFI. For the unlabeled bead control, we did not use amine-functionalized beads that were not labeled with TCPP because the autofluorescence of the unlabeled beads was interfering with calculating the compensation matrices correctly. Instead, we used non-functionalized polystyrene beads similar in make and size. In addition, we kept the unlabeled and TCPP-labeled beads in separate tubes. With both sets of beads in the same tube, we observed some transfer of TCPP to the unlabeled beads (data not shown), which caused a right-shift in the MFI that prohibited the correct calculation of the compensation matrix.

In summary, the critical points for achieving a high MFI of the TCPP-labeled beads are the reagent ratios used during the staining reaction, the time of the staining reaction, and the diameter of the unlabeled beads. In this work, the protocol described above afforded TCPP-stained beads that were appropriately sized for use in a flow cytometer and were sufficiently fluorescent for compensation. The TCPP-stained beads did not lose significant MFI at 4 °C for 300 days. The fluorescence spectrum of the TCPP beads was comparable to that of TCPP in solution. Moreover, the compensation matrices calculated with TCPP-labeled beads and stained A549 cells generated virtually identical sputum profiles in our assay, demonstrating the utility of these beads.

This protocol describes the preparation of TCPP compensation beads that were useful on the Navios EX instrument, but these same beads may not work on a different instrument with different sensitivity³⁶ and a different detector configuration²⁰. However, this work suggests ways of modulating the fluorescence through the bead size and reagent ratio. TCPP is only one of several fluorescent porphyrins that have demonstrated cancer cell selectivity in vitro and in vivo²^,³⁷^,³⁸^,³⁹. Not all fluorescent porphyrins may bind to the amine-functionalized polystyrene beads used in this protocol. In fact, the use of these beads is limited to porphyrins that contain a carboxylic acid. The differential staining of diverse cell populations in human tissue samples with porphyrins, as measured by flow cytometry, might yield insights into the early development of cancer, its progression, and its prognosis. The use of other porphyrins to stain human tissue samples and their analysis by flow cytometry, using appropriate control beads as compensation standards, is an attractive direction for further research.

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

References

Josefsen, L. B., Boyle, R. W. Unique diagnostic and therapeutic roles of porphyrins and phthalocyanines in photodynamic therapy, imaging and theranostics. Theranostics. 2 (9), 916-966 (2012).
Tsolekile, N., Nelana, S., Oluwafemi, O. S. Porphyrin as diagnostic and therapeutic agent. Molecules. 24 (14), 2669 (2019).
Gunaydin, G., Gedik, M. E., Ayan, S. Photodynamic therapy for the treatment and diagnosis of cancer-A review of the current clinical status. Frontiers in Chemistry. 9, 686303 (2021).
Berg, K., et al. Porphyrin-related photosensitizers for cancer imaging and therapeutic applications. Journal of Microscopy. 218, 133-147 (2005).
Kessel, D., Reiners, J. Light-activated pharmaceuticals: Mechanisms and detection). Israel Journal of Chemistry. 52 (8-9), 674-680 (2012).
Didamson, O. C., Abrahamse, H. Targeted photodynamic diagnosis and therapy for esophageal cancer: Potential role of functionalized nanomedicine. Pharmaceutics. 13 (11), 1943 (2021).
Harada, Y., Murayama, Y., Takamatsu, T., Otsuji, E., Tanaka, H. 5-Aminolevulinic acid-induced protoporphyrin IX fluorescence imaging for tumor detection: Recent advances and challenges. International Journal of Molecular Sciences. 23 (12), 6478 (2022).
Bochenek, K., Aebisher, D., Międzybrodzka, A., Cieślar, G., Kawczyk-Krupka, A. Methods for bladder cancer diagnosis – The role of autofluorescence and photodynamic diagnosis. Photodiagnosis and Photodynamic Therapy. 27, 141-148 (2019).
Iwaki, K., et al. Flow cytometry-based photodynamic diagnosis with 5-aminolevulinic acid for the detection of minimal residual disease in multiple myeloma. The Tohoku Journal of Experimental Medicine. 249 (1), 19-28 (2019).
Patriquin, L., et al. Early detection of lung cancer with meso tetra (4-carboxyphenyl) porphyrin-labeled sputum. Journal of Thoracic Oncology. 10 (9), 1311-1318 (2015).
Pan, L., et al. A brief introduction to porphyrin compounds used in tumor imaging and therapies. Mini Reviews in Medicinal Chemistry. 21 (11), 1303-1313 (2021).
Nishida, K., Tojo, T., Kondo, T., Yuasa, M. Evaluation of the correlation between porphyrin accumulation in cancer cells and functional positions for application as a drug carrier. Scientific Reports. 11 (1), 2046 (2021).
Lin, Y., Zhou, T., Bai, R., Xie, Y. Chemical approaches for the enhancement of porphyrin skeleton-based photodynamic therapy. Journal of Enzyme Inhibition and Medicinal Chemistry. 35 (1), 1080-1099 (2020).
Kou, J., Dou, D., Yang, L. Porphyrin photosensitizers in photodynamic therapy and its applications. Oncotarget. 8 (46), 81591-81603 (2017).
Wezgowiec, J., et al. Electric field-assisted delivery of photofrin to human breast carcinoma cells. The Journal of Membrane Biology. 246 (10), 725-735 (2013).
Palasuberniam, P., et al. Small molecule kinase inhibitors enhance aminolevulinic acid-mediated protoporphyrin IX fluorescence and PDT response in triple negative breast cancer cell lines. Journal of Biomedical Optics. 26 (9), 098002 (2021).
Kim, B., Bohandy, J. Spectroscopy of porphyrins. Johns Hopkins APL Technical Digest. 2 (3), 153-163 (1981).
Uttamlal, M., Sheila Holmes-Smith, A. The excitation wavelength dependent fluorescence of porphyrins. Chemical Physics Letters. 454 (4), 223-228 (2008).
Zhang, Y. Z., Kemper, C., Bakke, A., Haugland, R. P. Novel flow cytometry compensation standards: internally stained fluorescent microspheres with matched emission spectra and long-term stability. Cytometry. 33 (2), 244-248 (1998).
Monard, S. Building a spectral cytometry toolbox: Coupling fluorescent proteins and antibodies to microspheres. Cytometry. Part A. 101 (10), 846-855 (2022).
Byrd, T., et al. Polystyrene microspheres enable 10-color compensation for immunophenotyping of primary human leukocytes. Cytometry. Part A. 87 (11), 1038-1046 (2015).
Roederer, M. Compensation in flow cytometry. Current Protocols in Cytometry. , (2002).
Kabe, Y., et al. Porphyrin accumulation in mitochondria is mediated by 2-oxoglutarate carrier. The Journal of Biological Chemistry. 281 (42), 31729-31735 (2006).
Bederka, L. H., et al. Sputum analysis by flow cytometry; An effective platform to analyze the lung environment. PLoS One. 17 (8), e0272069 (2022).
. US6838248B2 – Compositions and methods for detecting pre-cancerous conditions in cell and tissue samples using 5, 10, 15, 20-tetrakis (carboxyphenyl) porphine Available from: https://patents.google.com/patent/US68248B2/en?oq=US+patent+6838248+B2 (2005)
. Method of using 5,10,15,20-tetrakis(carboxyphenyl)porphine for detecting cancers of the lung Available from: https://www.osti.gov/deopatents/biblio/7117152 (1992)
Grayson, M., et al. Quality-controlled sputum analysis by flow cytometry. Journal of Visualized Experiments. (174), e62785 (2021).
Anjali, K., Christopher, J., Sakthivel, A. Ruthenium-based macromolecules as potential catalysts in homogeneous and heterogeneous phases for the utilization of carbon dioxide. ACS Omega. 4 (8), 13454-13464 (2019).
Yadav, R., et al. Recent advances in the preparation and applications of organo-functionalized porous materials. 화학. 15 (17), 2588-2621 (2020).
. US7670799B2 – Method for making 5,10,15,10-tetrakis (carboxyphenyl) porphine (TCPP) solutions and composition compromising TCPP Available from: https://patents.google.com/patent/US7670799B2/en (2023)
Shimizu, N., et al. High-performance affinity beads for identifying drug receptors. Nature Biotechnology. 18 (8), 877-881 (2000).
Anderson, G. W., Zimmerman, J. E., Callahan, F. M. The use of esters of N-hydroxysuccinimide in peptide synthesis. Journal of the American Chemical Society. 86 (9), 1839-1842 (1964).
Hermanson, G. T. . Bioconjugate Techniques. , (2013).
El-Faham, A., Albericio, F. Peptide coupling reagents, more than a letter soup. Chemical Reviews. 111 (11), 6557-6602 (2011).
Hulspas, R., O’Gorman, M. R. G., Wood, B. L., Gratama, J. W., Sutherland, D. R. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry Part B. 76 (6), 355-364 (2009).
Hoffman, R. A. Standardization, calibration, and control in flow cytometry. Current Protocols in Cytometry. , (2005).
Ethirajan, M., Chen, Y., Joshi, P., Pandey, R. K. The role of porphyrin chemistry in tumor imaging and photodynamic therapy. Chemical Society Reviews. 40 (1), 340-362 (2011).
Beharry, A. A. Next-generation photodynamic therapy: New probes for cancer imaging and treatment. 생화학. 57 (2), 173-174 (2018).
El-Far, M., Pimstone, N. A comparative study of 28 porphyrins and their abilities to localize in mammary mouse carcinoma: Uroporphyrin I superior to hematoporphyrin derivative. Progress in Clinical and Biological Research. 170, 661-672 (1984).

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Bauta, W., Grayson, M., Titone, R., Rebeles, J., Rebel, V. I. Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry. J. Vis. Exp. (193), e65294, doi:10.3791/65294 (2023).