HepG2 cells (3.0 × 104/well) were seeded in black 96-well plates and kept in complete growth medium for 2 d before treatment. The third day, they were treated with 4.0 μg/mL PCSK9 and 4.0 μg/mL PCSK9 + peptides (0.5-50.0 µM), and vehicle (H2O) for 2 h with at 37 °C under 5% CO2 atmosphere. At the end of the treatments, the culture medium was replaced with 50.0 μl/well LDL-DyLight™ 550 working solution (Cayman Chemical Company, Ann Arbor, MI, US). The cells were additionally incubated for 2 h at 37 °C and then the culture medium was aspirated and replaced with PBS (100.0 μl/well). The degree of LDL uptake was measured using the Synergy H1 fluorescent plate reader from Biotek (excitation and emission wavelengths 540 and 570 nm, respectively).
Ldl dylight 550 working solution
Manufactured by Cayman Chemical
Sourced in United States
LDL-DyLight™ 550 working solution is a laboratory reagent designed for use in research applications. It is a ready-to-use solution containing a fluorescent dye that can be used to label and detect low-density lipoprotein (LDL) molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
6 protocols using ldl dylight 550 working solution
1
Quantification of PCSK9-mediated LDL Uptake
2
LDL Uptake Assay in HepG2 Cells
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HepG2 cells (3 × 104/well) were seeded in 96-well plates and kept in complete growth medium for 2 d before treatment. The third day, they were treated with T9 (160 and 320 μM) or P5 (10 μM), or vehicle (H2O) for 24 h. At the end of the treatment, the culture medium was replaced with 50 μl/well LDL-DyLight™ 550 working solution (Cayman Chemical Company, Ann Arbor, MI, US). The cells were additionally incubated for 2 h at 37 °C and then the culture medium was aspirated and replaced with PBS (100 μl/well). The degree of LDL uptake was measured using the Synergy H1 fluorescent plate reader from Biotek (excitation and emission wavelengths 540 and 570 nm, respectively).
3
Quantifying Cellular LDL Uptake
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HepG2 cells (2 × 104 cells per well) were seeded in black 96-well plates and cultured for more than 24 h. The following day, cells were treated with 4.0 μg/ml K9 alone and the tested compounds with the presence of 4.0 μg/ml of K9, and vehicle (PBS) for 8 h. At the end of the treatments, the culture medium was replaced with 50.0 μl/well LDL-DyLight™ 550 working solution (Cayman Chemical Company, US). The cells were additionally incubated for 16 h at 37 °C. Then the cells were washed with PBS one time, then added PBS with 100.0 μl/well. The degree of LDL uptake was measured using a plate reader (EnSpire, Perkin Elmer Corporation) at excitation wavelengths 540 nm and emission wavelengths 570 nm.
4
Measuring PCSK9-Mediated LDL Uptake
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HepG2 cells (3 × 104/well) were seeded in 96-well plates and kept in complete
growth medium for 2 days before treatment. On the third day, the cells
were washed with PBS and then starved overnight (O/N) in DMEM without
FBS and antibiotics. After starvation, they were treated with 4.0
μg/mL PCSK9, 4.0 μg/mL PCSK9 + diimidazole analogues,
and vehicle (H2O) for 2 h with at 37 °C under a 5%
CO2 atmosphere. At the end of the treatment, the culture
medium was replaced with 50 μL/well LDL-DyLight 550 working
solution (Cayman Chemical Company, Ann Arbor, MI, USA) prepared in
DMEM without FBS and antibiotics. The cells were additionally incubated
for 2 h at 37 °C, and then the culture medium was aspirated and
replaced with PBS (100 μL/well). The degree of LDL uptake was
measured using a Synergy H1 fluorescent plate reader (Winooski, VT,
USA) (excitation and emission wavelengths of 540 and 570 nm, respectively).
Fluorescent LDL uptake was finally assessed following optimized protocol.14 (link)
growth medium for 2 days before treatment. On the third day, the cells
were washed with PBS and then starved overnight (O/N) in DMEM without
FBS and antibiotics. After starvation, they were treated with 4.0
μg/mL PCSK9, 4.0 μg/mL PCSK9 + diimidazole analogues,
and vehicle (H2O) for 2 h with at 37 °C under a 5%
CO2 atmosphere. At the end of the treatment, the culture
medium was replaced with 50 μL/well LDL-DyLight 550 working
solution (Cayman Chemical Company, Ann Arbor, MI, USA) prepared in
DMEM without FBS and antibiotics. The cells were additionally incubated
for 2 h at 37 °C, and then the culture medium was aspirated and
replaced with PBS (100 μL/well). The degree of LDL uptake was
measured using a Synergy H1 fluorescent plate reader (Winooski, VT,
USA) (excitation and emission wavelengths of 540 and 570 nm, respectively).
Fluorescent LDL uptake was finally assessed following optimized protocol.14 (link)
5
Modulation of LDL Uptake in HepG2 Cells
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Experiments were performed as previously described. 35 HepG2 cells (3.0 x 10 4 well -1 ) were seeded in black 96-well plates and kept in complete growth medium for 2 d before treatment.
The third day, they were treated with either 4 μg/mL PCSK9, or 4 μg/mL PCSK9 with either tEGF-A (0.5-50 µM), TEX-S2_03 (0.05-5 µM), or TEX-S3_01 (0.05-50 µM), or vehicle (H2O) for 2 h with at 37 °C under 5% CO2 atmosphere. At the end of the treatments, the culture medium was replaced with LDL-DyLight™ 550 working solution (Cayman Chemical Company). The cells were additionally incubated for 2 h at 37 °C and then the culture medium was aspirated and replaced with PBS. The degree of LDL uptake was measured using the Synergy H1 fluorescent plate reader (excitation and emission wavelengths 540 and 570 nm, respectively). Data were the average ± s.d. of three independent experiments, each performed with three replicates, using GraphPad Prism. Statistical analyses were carried out by student ttest. P-values < 0.05 were considered to be significant.
The third day, they were treated with either 4 μg/mL PCSK9, or 4 μg/mL PCSK9 with either tEGF-A (0.5-50 µM), TEX-S2_03 (0.05-5 µM), or TEX-S3_01 (0.05-50 µM), or vehicle (H2O) for 2 h with at 37 °C under 5% CO2 atmosphere. At the end of the treatments, the culture medium was replaced with LDL-DyLight™ 550 working solution (Cayman Chemical Company). The cells were additionally incubated for 2 h at 37 °C and then the culture medium was aspirated and replaced with PBS. The degree of LDL uptake was measured using the Synergy H1 fluorescent plate reader (excitation and emission wavelengths 540 and 570 nm, respectively). Data were the average ± s.d. of three independent experiments, each performed with three replicates, using GraphPad Prism. Statistical analyses were carried out by student ttest. P-values < 0.05 were considered to be significant.
6
LDL Uptake Assay in HepG2 Cells
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HepG2 cells (3 × 104/well) were seeded in 96-well plates and kept in complete growth medium for 2 days before treatment. The third day, cells were washed with PBS and then starved overnight (O/N) in DMEM without FBS and antibiotics. After starvation, they were treated with 4.0 μg/mL PCSK9 and 4.0 μg/mL PCSK9 + P5 and P5-met peptides (50.0 µM), and vehicle (H2O) for 2 h with at 37 °C under 5% CO2 atmosphere. At the end of the treatment, the culture medium was replaced with 50 μL/well LDL-DyLight™ 550 working solution (Cayman Chemical Company, Ann Arbor, MI, USA) prepared in DMEM without FBS and antibiotics. The cells were additionally incubated for 2 h at 37 °C and then the culture medium was aspirated and replaced with PBS (100 μL/well). The degree of LDL uptake was measured using the Synergy H1 fluorescent plate reader (Winooski, VT, USA) (excitation and emission wavelengths 540 and 570 nm, respectively). Fluorescent LDL-uptake was finally assessed following optimized protocol [12 (link)].
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