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

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 properties^1,2,3. 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 death^4,5. 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 cancer^7,8. 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 agents^2,11. 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 compounds^{12,13,14,15,16}. 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.

1. 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.
2. 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.
3. Determine the required amount of sodium bicarbonate from Table 1 based on the amount of TCPP weighed in step 1.1.
4. 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.
5. Add the sodium bicarbonate to the 100 mL beaker containing purified water and isopropanol. Cover the solution with parafilm to protect from evaporation.
6. Place the solution from step 1.5 on a stir plate, and stir until dissolved (approx. 10 min)
7. Measure the pH to ensure that the sodium bicarbonate-containing solution from step 1.6 has a pH between 9 and 10.
8. 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.
9. 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.

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

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

1. 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

1. Add 4.3 mL of the 0.1 M MES buffer solution prepared in step 2 to a 15 mL polypropylene tube.
2. Vortex the amine-functionalized polystyrene bead suspension (10 µm, 2.5% w/v) for 60 s at maximum speed.
3. Add 288 µL of this freshly vortexed bead suspension to the MES buffer from step 4.1.
4. Vortex the MES/bead solution for 15 s at maximum speed.
5. Vortex the 1 mg/mL TCPP solution prepared in step 1 for 60 s at maximum speed.
6. Add 1.20 mL of this freshly vortexed TCPP stock solution to the MES/bead suspension from step 4.4.
7. Vortex the MES/bead/TCPP suspension for 15 s at maximum speed.
8. 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.

1. Add 20.0 mL of purified water to a new 50 mL conical tube.
2. Weigh out 200 mg of EDC HCl from step 3, and add it to the water (step 5.1).
3. 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.

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

1. 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).
2. Place the tube in an inverting rotator at 35 rpm for 16 h at room temperature and protected from light.
3. Centrifuge the tube at room temperature for 10 min at 1,000 × g.
4. Aspirate the supernatant, and resuspend the beads in 0.8 mL of Hanks' balanced salt solution (HBSS).
5. 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.

1. 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.
2. Label another tube as "TCPP positive beads."
3. Label another tube as "Rainbow beads."
4. Aliquot 300 µL of ice-cold HBSS into the tubes labeled with "TCPP negative beads" and "TCPP positive beads."
5. Add 500 µL of ice-cold HBSS into the tube labeled as "Rainbow beads."
6. 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."
7. 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.
8. Vortex the Rainbow bead suspension briefly at maximum speed, and add two drops of it into the "Rainbow beads" tube.
9. Keep all tubes on ice, covered, and protected from light.
10. 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.
11. 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).

1. 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).
2. Vortex the diluted bead suspension at maximum speed for 15 s.
3. Remove the plunger from a disposable 5 mL syringe.
4. Fit the syringe with a glass fiber tip filter (5 µm, 13 mm diameter).
5. Add 4 mL of HBSS to the syringe.
6. Add 0.5 mL of the vortexed diluted bead suspension (step 9.2).
7. Use the plunger to filter the suspension through the syringe/filter setup at approximately 2 drops/s.
8. Wash the beads by drawing 5 mL of fresh HBSS into the syringe through the filter at approximately 2 drops/s.
9. Push the HBSS out again into the waste container at approximately 2 drops/s.
10. To remove the beads from the filter, draw another 5 mL of fresh HBSS into the syringe through the filter.
11. Carefully remove the filter from the syringe.
12. Eject the bead suspension from the syringe into a 50 mL conical centrifuge tube.
13. Place the filter back on the syringe, and repeat steps 9.10-9.12 four more times. Then discard the filter and syringe.
14. 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.
15. Centrifuge the filtered bead suspensions for 10 min at 1,000 × g at room temperature.
16. Aspirate the supernatant of each 50 mL tube, and gently resuspend the beads, combining them in 0.5 mL of fresh HBSS.
17. Transfer the beads with a p1,000 micropipette to a new amber, glass, or polypropylene vial, and store at 4 °C.
18. 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.

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 s...

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 tissues^24,25,26. Our research on the flow cytometric analysis of human sputum^24,27 requires the...

Materials

Name	Company	Catalog Number	Comments
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 1011 amine 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