Excitation and emission scans were performed on PTI QuantaMaster Fluorimeter from Horiba using FelixGX software (v. 4). 300 μL of cell lysate or bacterial suspension were placed in special optical glass cuvettes with 4 mm path length (23–4.45-SOG-4, Starna Cell, Inc.). Measurements were taken at 25°C. Measurements were taken at 1 nm intervals with a 1 second integration time.
Pti quantamaster fluorimeter
Manufactured by Horiba
Sourced in United States
The PTI QuantaMaster Fluorimeter is a high-performance fluorescence spectroscopy instrument used for analyzing the optical properties of samples. It is designed to measure the intensity and wavelength of fluorescent emissions from various materials and compounds.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using pti quantamaster fluorimeter
1
Fluorescence Excitation and Emission Analysis
2
Fluorescence Lifetime Measurement Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Decay curves were obtained on a PTI QuantaMaster Fluorimeter from Horiba using FelixGX software (v. 4) and a whitelase SC450 pulsed laser using a repetition rate of 20MHz. 300 μL of cell lysate or bacterial suspension were placed in special optical glass cuvettes with 4mm path length (23–4.45-SOG-4, Starna Cell, Inc.). Measurements were taken at 25°C until peak photon count reached 10,000. Lifetimes were calculated from the decay cures using the data analysis package from FelixGX. All fluorescence lifetimes were calculated by fitting a mono-exponential decay. Selected decay curves are shown in (S3 Fig ).
3
Optimizing Fluorescent Protein Expression in HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were used to test the point mutations because the ultimate goal was to use the optimized fluorescent protein in mammalian cells. HEK293T are good model cell line to apply for this purpose because they are particularly easy to grow and transfect. The HEK293T cells [39 ] were a gift from Frank Graham at McMaster University. Cells were grown in DMEM complete media. Cells were transiently transfected using calcium phosphate [40 (link)] for 16 hours. Cells were allowed to grow for another 24 hours after media change before imaging or harvesting. For spectral analysis experiments, cells were grown and transfected in 10 cm dishes. 24 hours after transfection cells were washed three times with PBS and once with hypotonic lysis buffer (1.5 mM MgCl2, 100nM HEPES, pH = 7.4, and protease inhibitors). Cells were incubated in 1 mL hypotonic lysis buffer on ice for 10 mins before homogenization using a Potter homogenizer. The suspension was centrifuged at 200 g for 10 mins at 4°C, and the supernatant was used for spectral analysis on a PTI QuantaMaster Fluorimeter from Horiba using FelixGX software (v. 4).
4
Membrane Fluidity Changes in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasma membrane fluidity of HeLa cells was estimated by means of the fluorescence anisotropy of the hydrophobic probe TMA-DPH (1-4-trimethylammoniumphenil-6-phenil-1,3,5-hexatriene; Thermo Fisher Scientific). HeLa cells (70–80% confluence) were incubated with L. crispatus BC5, S. agalactiae, E. faecalis, or B. subtilis cells (5 × 107 CFU) for 1 h at 37°C with 5% CO2, then washed three times with PBS and re-suspended at a final concentration of 3 × 105 cells/mL. The absorbance of the cell suspension was kept lower than 0.15 at the excitation wavelength of TMA-DPH. A few microliters of TMA-DPH stock solution were added to the cell suspension in order to obtain a final probe concentration of 1 μM. Fluorescence anisotropy measurements were performed by using a PTI QuantaMaster fluorimeter (Photon Technology International, North Edison, NJ, United States) equipped with a temperature-controlled cell holder and Polaroid HNP’B polarizers. Temperature was kept at 25°C. Excitation and emission wavelengths were set at 360 and 430 nm, respectively.
5
Membrane Fluidity Measurements by Fluorescence Anisotropy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Steady-state fluorescence anisotropy was used to investigate the possible modifications induced by PBS or sugar solutions on the physico-chemical state of CT EB membranes. The membrane fluidity of HeLa cells and CT EBs was estimated by measuring fluorescence anisotropy of the hydrophobic probe TMA-DPH [1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate] (ThermoFisher Scientific, Waltham, MA). TMA-DPH is a lipophilic fluorophore that penetrates the membrane hydrophobic core, orienting perpendicularly to the membrane plane [36 (link)]. In case of an increased fluidity of the membrane, the TMA-DPH probe rotates to a greater extent, leading to a depolarization of the fluorescence emission and a decrease in the fluorescence anisotropy.
Anisotropy was measured after 2 h-incubation with sugar solutions (glucose, sucrose, and mannitol 5 mM) for 2 h, at 37 °C, with 5% CO2. A PBS solution without sugars was used as a control.
HeLa cells and EBs were suspended at a final concentration of 3 × 105 cells/mL and 5 × 104 cells/mL, respectively. For the anisotropy measurements, the samples were incubated with TMA-DPH and then analyzed by a PTI QuantaMaster fluorimeter (Photon Technology International, North Edison, NJ, USA) according to Parolin et al. [30 (link)].
Anisotropy was measured after 2 h-incubation with sugar solutions (glucose, sucrose, and mannitol 5 mM) for 2 h, at 37 °C, with 5% CO2. A PBS solution without sugars was used as a control.
HeLa cells and EBs were suspended at a final concentration of 3 × 105 cells/mL and 5 × 104 cells/mL, respectively. For the anisotropy measurements, the samples were incubated with TMA-DPH and then analyzed by a PTI QuantaMaster fluorimeter (Photon Technology International, North Edison, NJ, USA) according to Parolin et al. [30 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!