To determine miPB-IPC’s multipotency and their RPE cell differentiation (Figure S1 ), miPB-IPC were treated with combined supplements (including L-glutamine, Gentamicin sulfate-Amphotericin (GA-1000), and basic fibroblast growth factor) in the presence of retinal pigment epithelial growth media (Lonza) for 8 days, in 24-well tissue culture-treated plates, at 37 °C in 5% CO2. The differentiated cells were characterized by immunocytochemistry with RPE-specific markers such as mouse anti-human mAbs RPE 65, CRALBP, and claudin-19, along with rabbit anti-tight junction protein 1 (ZO-1) polyclonal Ab (Novus Biological, Littleton, CO, USA). Human primary RPE cells were purchased from Lonza and served as positive control. Isotype-matched IgG served as negative control for immunostaining. For functional analysis, the phagocytosis of fluorescence latex beads (Sigma, Saint Louis, MO, USA) were performed in differentiated RPE cells. The phagocytosis-associated surface marker CD36 was examined by flow cytometry. The level of CD36 expression was quantified by mean fluorescence intensity after analyzed with Kaluza software version 2.1 (Beckman Coulter).
Fluorescence latex beads
Manufactured by Merck Group
Sourced in United States
Fluorescence latex beads are a type of laboratory equipment used for various analytical and research applications. They are spherical particles composed of a polymer matrix that can be loaded with fluorescent dyes or other compounds. The beads are designed to emit light at specific wavelengths when excited by a light source, making them useful as labels, tracers, or probes in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Diff quik staining dye, by Merck Group (3 mentions)
Permount, by Merck Group (2 mentions)
Kaluza software version 2, by Beckman Coulter (1 mentions)
Axiovert imager m 2, by Zeiss (1 mentions)
Fluoromount g with dapi, by Thermo Fisher Scientific (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
7 protocols using fluorescence latex beads
1
Multipotency and RPE Differentiation of miPB-IPCs
2
Phagocytosis Assay for Retinal Pigment Epithelial Cells
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For phagocytosis assay confluent RPE cells on collagen I coated cover slips were used (please refer to Section 4.5 ). After treatment, phagocytosis staining was conducted as previously established [1 (link)]. Fluorescence latex beads (Sigma-Aldrich) were opsonized with photoreceptor outer segments prepared from porcine retinae. Opsonized beads were applied to the RPE cells and incubated for four hours. Cells were washed with media and PBS to remove excess beads. Cells were fixated with paraformaldehyde and mounted with Fluoromount-G™ with DAPI (Thermo Fisher Scientific). Fluorescence images were taken with Axiovert Imager M.2 and AxioVision Software (both Zeiss, Jena, Germany). The DAPI stained cell nuclei were counted per hand. Fluorescence beads were counted by self-programmed macro in Fiji (ImageJ2, https://imagej.net/software/fiji/downloads (accessed on 8 August 2022). For evaluation measured beads were set in relation to number of cell nuclei.
3
Phagocytic Activity Evaluation Protocol
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The phagocytosis was calculated according to (Yoshida, Kitao, 1991) with slight modification, for detail see (Van Doan, Hoseinifar, Sringarm, Jaturasitha, Yuangsoi, Dawood, Esteban, Ringø, Faggio, 2019) . Briefly, 200µL of leucocyte cell suspensions ( 2x 10 6 cells mL -1 ) were loaded on coverslips and incubated at room temperature for two hours. After incubation, the coverslips were washed with 3mL of RPMI-1640 to remove any non-adherent cells. Then, a solution of 200µL of fluorescence latex beads with a concentration of 2 x 10 7 of beads (mL -1 ) (Sigma-Aldrich, USA) was placed into each coverslip and incubated again at room temperature for 1.5 hours. The coverslips were then rewashed with 3mL of RPMI-1640 to remove any non-phagocytized bead. After washing, the coverslips were then fixed with methanol, and stained with Diff-Quik staining dye (Sigma-Aldrich, USA) for ten seconds. After staining, a wash of PBS (pH 7.4) removed any excessive stains. The washed coverslips were allowed to dry at room temperature and then attached to the slides with Permount (Merck, Germany). The number of phagocyte cells per 300 adhered cells was later counted microscopically. The phagocytic index (PI) and phagocytic rate (PR%) were calculated through the following equations: PI = (Number of phagocytized beads divided by the number of phagocytizing leukocytes) *100.
4
Phagocytic Latex Bead Assay
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Fluorescence latex beads of 1 μm diameter (Sigma‐Aldrich) were first preopsinized for 1 hour at 37°C at a ratio of 1:5 with fetal bovine serum (Gibco). Preopsinized beads were subsequently diluted in the different serum‐containing and serum‐free culture conditions for a final concentration of latex beads of 0.01% (Figure S2 A). Cells were in contact with medium and preopsonized latex beads for 1 hour at 37°C and subsequently fixed with cold 4% PFA solution for a posterior immunocytochemistry study for CD11b and Iba1 expression.
5
Phagocytic Activity Quantification
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The phagocytic activity assay was performed as described by van Doan et al. (2018) . 200 ml leukocyte suspensions 2x10 6 cells ml -1 were spread on coverslips and incubated for 2 h. Non-adherent cells were then removed by washing with RPMI 1640. 200 ml of fluorescence latex beads (Sigma) solution 2x10 7 of beads mL -1 was added on each coverslip and incubated for 1.5 h at room temperature. After incubation, the nonphagocytized beads were washed with RPMI 1640. The coverslips were then fixed with methanol and stained with Diff-Quick staining dye (Sigma) for 10 seconds. The excess stain was removed by washing with PBS (pH 7.4), and the number of phagocytized cells per 300 adhered cells was counted microscopically. The phagocytic index (PI) was determined as follows: PI=Average number of beads per cell divided by the number of phagocytizing cells.
6
Phagocytic Activity Evaluation Protocol
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The phagocytosis was calculated according to (Yoshida, Kitao, 1991) with slight modification, for detail see (Van Doan, Hoseinifar, Sringarm, Jaturasitha, Yuangsoi, Dawood, Esteban, Ringø, Faggio, 2019) . Briefly, 200µL of leucocyte cell suspensions ( 2x 10 6 cells mL -1 ) were loaded on coverslips and incubated at room temperature for two hours. After incubation, the coverslips were washed with 3mL of RPMI-1640 to remove any non-adherent cells. Then, a solution of 200µL of fluorescence latex beads with a concentration of 2 x 10 7 of beads (mL -1 ) (Sigma-Aldrich, USA) was placed into each coverslip and incubated again at room temperature for 1.5 hours. The coverslips were then rewashed with 3mL of RPMI-1640 to remove any non-phagocytized bead. After washing, the coverslips were then fixed with methanol, and stained with Diff-Quik staining dye (Sigma-Aldrich, USA) for ten seconds. After staining, a wash of PBS (pH 7.4) removed any excessive stains. The washed coverslips were allowed to dry at room temperature and then attached to the slides with Permount (Merck, Germany). The number of phagocyte cells per 300 adhered cells was later counted microscopically. The phagocytic index (PI) and phagocytic rate (PR%) were calculated through the following equations: PI = (Number of phagocytized beads divided by the number of phagocytizing leukocytes) *100.
7
Measurement of Phagocytic Activity
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Phagocytosis activity was measured via the procedure specified in Yoshida and Kitao [102] . Briefly, 200µL of leucocyte cell suspensions (2 x 10 6 cells mL -1 ) were loaded on coverslips and incubated at room temperature for two hours. After incubation, the coverslips were washed with 3mL of RPMI-1640 to remove any non-adherent cells.
Then, a solution of 200µL of fluorescence latex beads with a concentration of 2 × 10 7 of beads (mL -1 ) (Sigma-Aldrich, USA) was placed into each coverslip and incubated again at room temperature for 1.5 hours. The coverslips were then rewashed with 3mL of RPMI-1640 to remove any non-phagocytized bead. After washing, the coverslips were then fixed with methanol, and stained with Diff-Quik staining dye (Sigma-Aldrich, USA) for ten seconds. After staining, a wash of PBS (pH 7.
Then, a solution of 200µL of fluorescence latex beads with a concentration of 2 × 10 7 of beads (mL -1 ) (Sigma-Aldrich, USA) was placed into each coverslip and incubated again at room temperature for 1.5 hours. The coverslips were then rewashed with 3mL of RPMI-1640 to remove any non-phagocytized bead. After washing, the coverslips were then fixed with methanol, and stained with Diff-Quik staining dye (Sigma-Aldrich, USA) for ten seconds. After staining, a wash of PBS (pH 7.
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