Cellular Thermoprobe for Fluorescence Ratio (cat no FDV-0005L)

Price: $400.00
  • Item #: FDV-0005L
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Cellular Thermoprobe for Fluorescence Ratio
Cat. No.: FDV-0005
Size: 3 X 200µg
Storage: RT, Dark Storage 

 
 

Cellular Thermoprobe for Fluorescence Ratio

Product Background

Cellular Thermoprobe for Fluorescence Ratio is an innovative new tool that can provide unprecedented scientific insight.

Cellular Thermoprobe for Fluorescence Ratio is a fluorescent polymeric thermometer for living cells. It diffuses throughout  cells and gives information about intracellular temperature distribution by fluorescence ratio. Cellular Thermoprobe for Fluorescence Ratio can be delivered into cells without microinjection. In addition, it does not require Fluorescence Lifetime for measuring, making it easy-to-use. With its cell permeability, Cellular Thermoprobe for Fluorescence Ratio is applicable to both adherent and suspension cells. It enables researchers to distinguish intracellular regional temperatures at the organelle level in cultured mammalian cells. 

Fluorescence Ratio in living HEK293T cells.

Confocal fluorescence image (left) and fluorescence image at the emission of 490-530 nm (middle), and fluorescence image at the emission of 570-610 nm of the "Cellular Thermoprobe for Fluorescence Ratio" in HEK293T cells. Bar:20μm

Description

  • Catalog Number: FDV-0005
  • Lot Number: see vial label
  • Size: 200ug or 200ug x 3
  • Average Molecular Weight: 20,300
  • Purity: >99%
  • Appearance: Yellow powder
  • Solubility: Soluble in water
  • Spatial Resolution: 240 nm
  • Temperature Resolution: 0.01-0.25℃
  • License: This product is licensed by Tokyo University and KIRIN Co., Ltd

 

Chemical Structure

Features

•Average Molecular Weight: 20,300
•Purity: >99%
•Appearance: Yellow powder
•Solubility: Soluble in water
•Spatial Resolution: 240 nm

•Temperature Resolution: 0.01-0.25℃
•License: This product is licensed by Tokyo University and KIRIN Co., Ltd

Reconstitution and Storage

  • Shipping: Shipped at ambient temperature
  • Storage: Store at ambient temperature (powder). For reconstituted solution, store at +4℃. Protect from light.

Reconstitution

  1. Before open the top, spin the vial down briefly.
  2. Reconstitute 200ug powder of Cellular Thermoprobe for Fluorescence Ratio in 20ul of ultrapure water*1.
  3. Dissolve it completely by vortex or tapping.
  4. Store the stock solution (1% w/v) at +4℃. Protect from light. Note that the stock solution needs to be incubated at +4℃ at least overnight before proceeding to experiment to obtain full extension of the polymer.

*1 Ionic solutions such as DMEM and PBS inhibit the incorporation of Cellular Thermoprobe for Fluorescence Ratio. If you find poor solubility, put it on ice for a while until it dissolve.

Representative absorption and fluorescence spectra of DBThD-IA and BODIPY-IA.

The absorption spectra were measured in acetonitrile (black). The fluorescence spectra were measured with excitation at 458 nm in ethyl acetate (red), acetonitrile (orange) and methanol (blue).

Optimal excitation and emission will be determined by your own. As an example, excitation at 458 nm and emission1 at 490-530 nm, and emission2 at 570-610 nm would work well.

How to generate calibration curve*1

  1. Dilute 1ul of Cellular Thermoprobe for Fluorescence Ratio in water (1% w/v) with cell extract (20-100 ul).
  2. Put the solution on a glass bottom dish.
  3. Set the temperature of the stage heater at the lowest you can (e.g. 25℃).
  4. Measure the fluorescence ratio after the medium temperature becomes steady.
  5. Adjust the medium temperature at your choice (e.g. 26℃).
  6. Measure the fluorescence ratio after the medium temperature becomes steady.
  7. Repeat step 5-6 until reaching the maximum temperature of the stage heater.
  8. Plot the fluorescence ratio against temperature to obtain a calibration curve. Estimate the temperature of your sample based on the calibration curve.

*1 Calibration curve can be also generated by Spectrofluorometer or Fluorescence Plate Reader equipped with temperature control.

Introduction of Cellular Thermoprobe for Fluorescence Ratio into Suspension Cells

  1. Collect the suspension cells by centrifugation at 400 x g for 3 min and wash it with 1 ml of a 5 % glucose solution and centrifuge it.
  2. Remove the supernatant.
  3. Resuspend the cell pellets in a 5 % glucose solution at a density of 1 x 106 cells/ml.
  4. Add Cellular Thermoprobe for Fluorescence Ratio in water (1% w/v) to a 20-100 fold*1 volume of cell suspension.
  5. Incubate the cells at 25℃ for 10 min.
  6. Centrifuge it and remove supernatant, and add 1ml PBS.
  7. Centrifuge it and remove supernatant, and resuspend in PBS.
  8. For the fluorescence imaging, approximately 10l of the cell suspension is dropped onto a coverslip and observe it immediately*2.

*1 Optimal dilution rate of Cellular Thermoprobe for Fluorescence Ratio depends on cell types.
*2 Set the appropriate temperature (e.g. 32 ~ 33℃) in a microscope cage incubation chamber based on your calibration curve and/or experimental condition.

Introduction of Cellular Thermoprobe for Fluorescence Ratio into Adherent Cells

  1. Prepare the cells at the 30 to 50 % confluency on glass bottom dish or equivalent.
  2. Remove the medium and wash with a 5 % glucose solution*1.
  3. Add 0.01-0.05 w/v%*2 of Cellular Thermoprobe for Fluorescence Ratio in 5 % glucose solution*1*3.
  4. Incubate the cells at 25℃ for 10 min.
  5. Wash the cells with PBS three times.
  6. Add phenol red-free culture medium and measure the fluorescence with appropriate temperature in a microscope cage incubation chamber*4.

*1 In the case that the dissociation of the adherent cells were observed in 5% glucose solution, 5% glucose solution with 0.1 – 0.3 mM CaCl2 may improve it.
*2 Optimal dilution rate of Cellular Thermoprobe for Fluorescence Ratio depends on cell types.
*3 The volume of the solution depends on the culture dish type. But we assume that minimum amount of volume (50-100 l) is sufficient to measure the cellular temperature.
*4 Set the appropriate temperature (e.g. 32 ~ 33℃) in a microscope cage incubation chamber based on your calibration curve and/or experimental condition.
Note: Above methods (Reconstitution, Preparation of cell extract for calibration curve, How to generate calibration curve and introduction) should be optimized depending on the cell type and organisms you use.

Example of Calibration Curve in MOLT-4 cells

 

Fluorescence response (closed, left axis) and temperature resolution (open, right axis) in MOLT-4 cells. The temperature resolution of Cellular Thermoprobe for Fluorescence Ratio was 0.01 – 0.25℃ in the temperature range between 25 and 44℃.

Reference

1) Uchiyama S, et. al, Analyst, 2015, 140, 4498-4506

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