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Inorganic phosphate nanorods are a novel fluorescent label in cell biology.

Patra CR, Bhattacharya R, Patra S, Basu S, Mukherjee P, Mukhopadhyay D - J Nanobiotechnology (2006)

Bottom Line: We report the first use of inorganic fluorescent lanthanide (europium and terbium) ortho phosphate [LnPO4.H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology.These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC), 786-O cells, or renal carcinoma cells (RCC).The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS), differential interference contrast (DIC) microscopy, confocal microscopy, and transmission electron microscopy (TEM).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Mayo Clinic, Rochester, Minnesota, USA. patra.chittaranjan@mayo.edu

ABSTRACT
We report the first use of inorganic fluorescent lanthanide (europium and terbium) ortho phosphate [LnPO4.H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology. These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC), 786-O cells, or renal carcinoma cells (RCC). The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS), differential interference contrast (DIC) microscopy, confocal microscopy, and transmission electron microscopy (TEM). At concentrations up to 50 microg/ml, the use of [3H]-thymidine incorporation assays, apoptosis assays (TUNEL), and trypan blue exclusion illustrated the non-toxic nature of these nanorods, a major advantage over traditional organic dyes.

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TUNEL assay apoptosis of HUVEC. First row: positive control experiment, second row: untreated control experiment, third row: HUVEC treated with EuPO4·H2O at 50 μg/ml for 20 h of incubation at 37°C and fourth row: HUVEC treated with TbPO4·H2O at 50 μg/ml for 24 h of incubation at 37°C. TUNEL assay apoptosis of HUVEC using camptothecin (4 h incubation at 37°C) as positive inducer (First row). A: TMR red -stained nuclei of HUVEc appear in red color due to presence of apoptotic cells, A1: The DAPI-stained nuclei appear in blue and A2: merged picture of A and A1. First Column: The nuclei of HUVEC were stained with TMR red (B-D), red staining was not observed due to absence of no apoptotic cells. Second column: The DAPI-stained nuclei appear in blue (B1-D1), and Third column: merged picture of first and second column (B2-D2).
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Figure 9: TUNEL assay apoptosis of HUVEC. First row: positive control experiment, second row: untreated control experiment, third row: HUVEC treated with EuPO4·H2O at 50 μg/ml for 20 h of incubation at 37°C and fourth row: HUVEC treated with TbPO4·H2O at 50 μg/ml for 24 h of incubation at 37°C. TUNEL assay apoptosis of HUVEC using camptothecin (4 h incubation at 37°C) as positive inducer (First row). A: TMR red -stained nuclei of HUVEc appear in red color due to presence of apoptotic cells, A1: The DAPI-stained nuclei appear in blue and A2: merged picture of A and A1. First Column: The nuclei of HUVEC were stained with TMR red (B-D), red staining was not observed due to absence of no apoptotic cells. Second column: The DAPI-stained nuclei appear in blue (B1-D1), and Third column: merged picture of first and second column (B2-D2).

Mentions: To investigate whether uptake of these nanorods induce apoptosis, we assayed endothelial cells treated with LnPO4.H2O nanorods using two apoptotic methods: (i) fluorescence microscopy using the In Situ Cell Death Detection Kit, TMR red (Roche, Cat. No.#12 156 792 910) and (ii) flow cytometry using Annexin V-FITC Apoptosis Detection Kit (Biovision, Cat. No. K101-100). The TUNEL assay detects apoptosis-induced DNA fragmentation through a quantitative fluorescence assay and was performed according to the manufacturer's instructions. In tunnel assay, the positive control apoptosis has been induced in cells using camptothecin (~2.5 mM) for 4 h of incubation (Fig. 9(A-A2)). The red-colored (TMR red-stained nuclei) apoptotic cells (Fig. 9A) were visualized under a microscope, counted (6 fields per sample), and photographed using a digital fluorescence camera. The DAPI-stained nuclei appeared blue in Fig. 9.A1 and Fig. 9.A2 shows the merged images of TMR- and DAPI-stained cells. The results of the TUNEL assay for the untreated control HUVEC and HUVEC cells treated with LnPO4·H2O nanorods are shown in Fig. 9B–D. In the first column (B-D) of Figure 9, no nuclei of TMR red-stained HUVEC cells were detected due to the absence of apoptotic cells. Blue DAPI-stained nuclei are in the second column (B1-D1) and the third column (B2-D2) shows the merged images. There was no difference in the number of apoptotic cells (~0%) detected in the untreated control experiment (First row: B, B1 and B2) nor cells treated with EuPO4·H2O nanorods (second row: C, C1 and C2) and TbPO4·H2O nanorods (third row: D, D1 and D2). The results of Fig. 6 and Fig. 9 clearly indicate that these nanorods were not toxic to endothelial cells. Similarly, flow cytometry analysis yielded no difference in the number of apoptotic cells bewteen untreated controls and nanoparticle-treated (data not shown).


Inorganic phosphate nanorods are a novel fluorescent label in cell biology.

Patra CR, Bhattacharya R, Patra S, Basu S, Mukherjee P, Mukhopadhyay D - J Nanobiotechnology (2006)

TUNEL assay apoptosis of HUVEC. First row: positive control experiment, second row: untreated control experiment, third row: HUVEC treated with EuPO4·H2O at 50 μg/ml for 20 h of incubation at 37°C and fourth row: HUVEC treated with TbPO4·H2O at 50 μg/ml for 24 h of incubation at 37°C. TUNEL assay apoptosis of HUVEC using camptothecin (4 h incubation at 37°C) as positive inducer (First row). A: TMR red -stained nuclei of HUVEc appear in red color due to presence of apoptotic cells, A1: The DAPI-stained nuclei appear in blue and A2: merged picture of A and A1. First Column: The nuclei of HUVEC were stained with TMR red (B-D), red staining was not observed due to absence of no apoptotic cells. Second column: The DAPI-stained nuclei appear in blue (B1-D1), and Third column: merged picture of first and second column (B2-D2).
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Figure 9: TUNEL assay apoptosis of HUVEC. First row: positive control experiment, second row: untreated control experiment, third row: HUVEC treated with EuPO4·H2O at 50 μg/ml for 20 h of incubation at 37°C and fourth row: HUVEC treated with TbPO4·H2O at 50 μg/ml for 24 h of incubation at 37°C. TUNEL assay apoptosis of HUVEC using camptothecin (4 h incubation at 37°C) as positive inducer (First row). A: TMR red -stained nuclei of HUVEc appear in red color due to presence of apoptotic cells, A1: The DAPI-stained nuclei appear in blue and A2: merged picture of A and A1. First Column: The nuclei of HUVEC were stained with TMR red (B-D), red staining was not observed due to absence of no apoptotic cells. Second column: The DAPI-stained nuclei appear in blue (B1-D1), and Third column: merged picture of first and second column (B2-D2).
Mentions: To investigate whether uptake of these nanorods induce apoptosis, we assayed endothelial cells treated with LnPO4.H2O nanorods using two apoptotic methods: (i) fluorescence microscopy using the In Situ Cell Death Detection Kit, TMR red (Roche, Cat. No.#12 156 792 910) and (ii) flow cytometry using Annexin V-FITC Apoptosis Detection Kit (Biovision, Cat. No. K101-100). The TUNEL assay detects apoptosis-induced DNA fragmentation through a quantitative fluorescence assay and was performed according to the manufacturer's instructions. In tunnel assay, the positive control apoptosis has been induced in cells using camptothecin (~2.5 mM) for 4 h of incubation (Fig. 9(A-A2)). The red-colored (TMR red-stained nuclei) apoptotic cells (Fig. 9A) were visualized under a microscope, counted (6 fields per sample), and photographed using a digital fluorescence camera. The DAPI-stained nuclei appeared blue in Fig. 9.A1 and Fig. 9.A2 shows the merged images of TMR- and DAPI-stained cells. The results of the TUNEL assay for the untreated control HUVEC and HUVEC cells treated with LnPO4·H2O nanorods are shown in Fig. 9B–D. In the first column (B-D) of Figure 9, no nuclei of TMR red-stained HUVEC cells were detected due to the absence of apoptotic cells. Blue DAPI-stained nuclei are in the second column (B1-D1) and the third column (B2-D2) shows the merged images. There was no difference in the number of apoptotic cells (~0%) detected in the untreated control experiment (First row: B, B1 and B2) nor cells treated with EuPO4·H2O nanorods (second row: C, C1 and C2) and TbPO4·H2O nanorods (third row: D, D1 and D2). The results of Fig. 6 and Fig. 9 clearly indicate that these nanorods were not toxic to endothelial cells. Similarly, flow cytometry analysis yielded no difference in the number of apoptotic cells bewteen untreated controls and nanoparticle-treated (data not shown).

Bottom Line: We report the first use of inorganic fluorescent lanthanide (europium and terbium) ortho phosphate [LnPO4.H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology.These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC), 786-O cells, or renal carcinoma cells (RCC).The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS), differential interference contrast (DIC) microscopy, confocal microscopy, and transmission electron microscopy (TEM).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Mayo Clinic, Rochester, Minnesota, USA. patra.chittaranjan@mayo.edu

ABSTRACT
We report the first use of inorganic fluorescent lanthanide (europium and terbium) ortho phosphate [LnPO4.H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology. These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC), 786-O cells, or renal carcinoma cells (RCC). The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS), differential interference contrast (DIC) microscopy, confocal microscopy, and transmission electron microscopy (TEM). At concentrations up to 50 microg/ml, the use of [3H]-thymidine incorporation assays, apoptosis assays (TUNEL), and trypan blue exclusion illustrated the non-toxic nature of these nanorods, a major advantage over traditional organic dyes.

No MeSH data available.


Related in: MedlinePlus