SKU: 55206806848

Fluo-8免洗细胞钙流检测试剂盒

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Description

Fluo-8免洗细胞钙流检测试剂盒Product Specification Synonyms Fluo 8 No Wash Calcium Flux Assay Kit Stability & Storage The unopened buffer solution should be stored at 4. Before use, it needs to be taken out and allowed to equilibrate at room temperature for 30 minutes, then inverted and mixed several times. The aliquoted buffer solution or 1x working solution can be stored at room temperature (22) for 1 week. The Fluo 8 reagent should be stored at 20 or 80 and will remain stable

Product Specification


Synonyms Fluo-8 No Wash Calcium Flux Assay Kit
Stability & Storage

The unopened buffer solution should be stored at 4℃. Before use, it needs to be taken out and allowed to equilibrate at room temperature for 30 minutes, then inverted and mixed several times. The aliquoted buffer solution or 1x working solution can be stored at room temperature (22℃) for 1 week. The Fluo-8 reagent should be stored at -20℃ or -80℃ and will remain stable for 1 year.

Background

Many vital intracellular biological reactions involve calcium flux. Detecting transient changes in intracellular calcium flux is crucial for studying intracellular biological processes and developing targeted drugs. The calcium-specific fluorescent dye method is the most commonly used technique for detecting intracellular calcium flux.
As a new generation calcium flux detection dye, Fluo-8 improves cellular loading and calcium response while maintaining the spectral wavelengths of Fluo-3 and Fluo-4 (Ex/Em ≈ 490/520 nm). Fluo-8 AM can be loaded into cells at room temperature, whereas Fluo-3 AM and Fluo-4 AM require 37°C for cell loading. Additionally, Fluo-8 AM is twice as bright as Fluo-4 AM and four times brighter than Fluo-3 AM.
In cellular loading and calcium flux analysis, calcium flux dyes often require cell washing, which significantly increases workflow during detection. Since some cells may detach during washing, this also introduces variability in the analysis. Therefore, no-wash dyes and methods represent a current trend in development.
To reduce dye efflux from cells, the anion transport protein inhibitor probenecid needs to be added to both the dye loading solution and subsequent analysis solutions.

Components

The contents of the kit include Fluo-8 reagent and buffer solution, which are separately filled in 1 ml spiral tubes and 10 ml or 100 ml brown bottles. The specifications are as follows:

Size

Fluo-8 regent

Buffer

The number of detectable wells in a 96-well plate

The number of detectable wells in a 384-well plate

100tests

100μl

10ml

200 wells

1,000 wells

1000tests

1ml

100ml

2,000 wells

10,000 wells

10000tests

10x1ml

10x100ml

20,000 wells

100,000 wells

Protocol

1. Cell Preparation
a) Digest cells cultured in normal medium with trypsin, add complete medium, count the cells, take the required total number of cells, and centrifuge. The following are suggested quantities for a 96-well plate; adjust accordingly for a 384-well plate. For a 96-well culture plate, it is generally recommended to use approximately 20,000 cells per well.
b) Resuspend the centrifuged cells in Hank’s salt solution or a similar phenol red-free culture medium supplemented with 1% fetal bovine serum. Then seed the cells into the culture plate at 50μl per well. Culture the cells for 3-5 hours until adherence or overnight.

2. Dye Loading
a) Take out the buffer and equilibrate at room temperature for 30 minutes, inverting and mixing several times. If the buffer is labeled as a 10x concentrate, dilute it to a 1x working buffer using deionized water.
b) Take out the Fluo-8 reagent and equilibrate at room temperature for 5-10 minutes, then centrifuge to collect the contents at the bottom of the tube. If the reagent is not used all at once, it can be aliquoted and stored at -20°C to minimize freeze-thaw cycles. Keep protected from light.
c) Calculate the required volume of dye loading solution based on 50μl per well. Add the Fluo-8 reagent to the 1x working buffer and mix thoroughly. The recommended starting dilution for Fluo-8 reagent is 1:100 in the working buffer, which can be adjusted based on cell type and requirements.
d) Calculate the required volume of probenecid stock solution and add it to the Fluo-8 working buffer, mixing well to prepare the loading solution.
e) Aspirate the culture medium from the wells of the culture plate and add the loading solution at 50μl per well. Gently shake for 10 seconds, cover, and incubate at 37°C for 1 hour.

3. Intracellular Calcium Flux Detection
a) Prepare compounds and agonists: It is recommended to use at least 6 concentration gradients for agonists. Include control wells without agonists or compounds in each reaction plate. The volume for agonist/compound plates is 100-200μl per well.
b) Prepare specialized pipette tip boxes compatible with the detection instrument.
c) Prepare the detection buffer: The detection buffer is Hank’s salt buffer supplemented with probenecid.
d) Aspirate the dye loading solution from the wells of the culture plate and add 100μl of detection buffer per well.
e) Set up the instrument detection program and relevant parameters, with excitation/emission wavelengths at 490/525nm. Read every 1-3 seconds for 2 minutes continuously.
f) Take a baseline reading before adding agonists/compounds.
g) Set up the corresponding positions of the agonist/compound plate wells and tips relative to the experimental cells.
h) Initiate the detection program for adding agonists/compounds and collect experimental data.
i) Perform data analysis using the (F-F0)/F0 *100% method or other validated methods.

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SKU: 55206806848

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C. Seiden
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Convenient Accessibility
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Bice
Whiting, US
★★★★★ 5
The Christmas movie no TV stations ever seem to show during the holidays
Since Amazon lumps the reviews for all the various releases of this movie together, I want to be clear that this review is for the single disc, 132 minute blu-ray version with the cover picture of smiling man made of bricks with the word "Brazil" flying out of the top of his head. For those who don't know the history of this movie - there are at least three different versions. It was originally released in Europe as a nearly two and half hour long film. The studio who financed it didn't like that version because it A. was too long, B. was too uncommercial and C. had a dark, depressing ending. The contract they had with Gilliam stipulated that the movie had to be less than 2 hours and 15 minutes, so it could have multiple prime-time showings in theaters. They suggested he cut the ending. Gilliam refused, so the studio took it upon themselves to create the "Love Conquers All" version, which chopped out almost an hour of footage - they only kept the parts that showed the main character Sam in a heroic light, anything that contributed to the Sam/Jill love story and anything that featured Robert DiNero. And, of course, the ending was changed to a happy ending by removing the final scene. Also, to make the plot easier to follow, they used some alternate takes, some deleted footage and hired voice actors to overdub dialog. Unsurprisingly, Gilliam didn't want that version released, so he finally edited the movie down to 2 hours and 12 minutes, kept the dark ending and made a few other minor changes and the studio was contractually obligated to release that version to theaters in the United States. It's this US version that appears on the blu-ray, with absolutely no additional features (not even the Gilliam commentary that's included on most other releases of the film). Annoyingly, they also added advertisements for other movies to the main menu screen. But I'm still happy to have this version, because it's the first time I've seen the "US edit" since originally seeing the film in a theater on the campus of Penn State back in my college days. I can't really write an unbiased review of the movie itself, because from that first viewing I became obsessed with both Brazil and Terry Gilliam's films in general. I bought all his movies on VHS as soon as they came out and eventually replaced them with DVDs. I bought the 3-DVD Criterion "Final Cut" version of Brazil back when it was an expensive new release. I was surprised that it included scenes I'd never seen before - I think it's based mostly on the European version, with some additional tweaks by Gilliam. It also included the "Love Conquers All" version of the movie and a ton of bonus features. Sadly though, it wasn't animorphic, so on my widescreen TV it put black letterboxing bars on all four sides, shrinking the picture down to about 60% of the screen. This blu-ray was exactly what I was looking for. While it's missing a few (non-essential) scenes, it's the version of the movie that I "grew up" with and is presented in full 16X9 animorphic widescreen. It's the best looking version I've seen since originally seeing it in the theater. For example, I never really noticed the makeup on Katherine Hellmond at the beginning of the movie to make her look older (pre-surgery), but it's clearly visible on the blu-ray. I've always felt bad for one actress during the explosion scene in the restaurant because it looks like she really does get hit by some of the flying debris - the blu-ray is maybe a little too clear there, because now I can tell it's just a dressed-up mannequin. As for the movie itself - it's one of those films you'll either love or hate. Its alternate-reality, retro-futuristic look makes it seem nearly as fresh and amazing today as it did when it first came out. And the background plot of a government bureaucracy turning a country into a surveillance state in response to a perceived terrorist threat - I know it was based on things happening in England at the time, but it's like Gilliam had a crystal ball and looked 30 years into the United States' future. The alternating between "reality" and dream sequences and the general Gilliam quirkiness will probably put a lot of people off, but it's what drew me to this movie in the first place. If you become fanatical about the movie like I am, you'll need both this blu-ray and the Criterion release. To fully experience the film you need to see every version (even the Love Conquers All version) because each one includes bits that aren't in any of the others. If you do decide to watch Brazil and find that you like it, give some of Gilliam's more recent movies a try, like The Imaginarium of Dr. Parnassis and The Zero Theorem. Really, if you like odd, out-of-the-mainstream movies that you'll be thinking about for days after you see them, you can't go wrong with pretty much any Terry Gilliam film.
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★★★★★ 5
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