Published: March 24th, 2023
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 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.
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 death
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.
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.......
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.......
|Amber plastic vials, 2 mL, U- bottom, polypropylene
|Research Products International
|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
|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
|with appropriate rotor
|Disposable polystyrene bottle with cap, 150 mL
|EDC (N- (3- dimethylaminopropyl)- N'- ethylcarbodiimide hydrochloride), ≥98%
|CAS No: 25952-53-8
|FlowJo Single Cell Analysis Software (v10.6.1)
|Glass coverslips, 22 x 22 mm
|Glass fiber syringe filters (Finneran, 5 µm, 13 mm diameter)
|Glass microscope slides, 275 x 75 x 1 mm
|Hanks Balanced Salt Solution (HBSS)
|Isopropanol, ACS grade
|Mechanical pipette, 1 channel, 100-1000 uL with tips
|MES (22- (N- mopholino)- N'- ethanesulfonic acid, hemisodium salt
|CAS No: 117961-21-4
|Navios EX flow cytometer
|Olympus BX-40 microscope with DP73 camera and 40X objective with cellSens software
|Pasteur pipettes, glass, 5.75"
|pH meter (UB 10 Ultra Basic)
|Pipette controller (Drummond)
|Plastic Syringe, 5 mL
|Polystyrene Particles (non-functionalized), SPHERO, 2.5% w/v, 8.0-12.9 µm
|Polypropylene tubes, 15mL, conical
|Polystyrene tubes, round bottom
|Rainbow Beads (Spherotech URCP-50-2K)
|Serological pipettes, disposable - 10 mL
|Serological pipettes, disposable - 25 mL
|Sodium bicarbonate (NaHCO3)
|CAS No: 144-55-8
|TCPP (meso-tetra(4-carboxyphenyl)porphine) Frontier Scientific
|CAS No: 14609-54-2
|Tecan Spark Plate Reader (or similar)
|Tecan Life Sciences
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