| Description | Free Fatty Acids (FFA), also known as Non-Esterified Fatty Acids (NEFA), are primarily produced by the hydrolysis of neutral fats. They are intermediate products in fat metabolism, involved in cell proliferation, inflammatory responses, and hormone regulation. FFA can also act as signaling moleculesFree Fatty Acids (FFA), also known as Non-Esterified Fatty Acids (NEFA), are primarily produced by the hydrolysis of neutral fats. They are intermediate products in fat metabolism, involved in cell proliferation, inflammatory responses, and hormone regulation. FFA can also act as signaling molecules with various physiological functions. Free fatty acids are closely related to lipid metabolism, glucose metabolism, and endocrine function. Their concentration is an important physiological and biochemical indicator, serving as an auxiliary parameter for disease evaluation and diagnosis, and also reflecting quality changes during food storage.Detection Principle: FFAs combine with copper ions to form copper soaps, which are soluble in chloroform. The copper ions can then react with a chromogenic solution to form a purplish-red complex. This product has a characteristic absorption peak at 550 nm. The FFA content can be quantified by measuring the change in absorbance.Detection Range: 0.0313 - 2 mMSensitivity: 0.0156 mMApplicable Samples: Serum (plasma), animal/plant tissues, cells, bacteria.A1492746Component48T96TStorageA1492746ACu Reagent6 mL12 mL2-8℃. Store in the dark.A1492746BChromogen15 mL30 mL2-8℃. Store in the dark.A1492746CStandard (16.41 mg Palmitic Acid)1 EA1 EA2-8℃. Store in the dark.User-Prepared Instruments and ReagentsMicroplate reader or visible spectrophotometer (capable of measuring absorbance at 550 nm)Incubator, Ice maker, Low-temperature centrifuge96-well plate or micro glass cuvettes, Adjustable pipettes and tipsHomogenizer (for tissue samples)Glass bottle (for preparing extraction buffer)n-Heptane, Anhydrous methanol, ChloroformExperimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsExtraction Buffer (Self-prepared)In a glass bottle, mix Chloroform : n-Heptane : Anhydrous Methanol = 28 : 21 : 1. Cap tightly and mix well.Store at 4°C protected from light.Cu ReagentReady-to-use; equilibrate to room temperature before use; mix well before use.Store at 4°C protected from light.ChromogenReady-to-use; equilibrate to room temperature before use.Store at 4°C protected from light.StandardBefore use, dissolve contents in 1 mL of Extraction Buffer to obtain a 64 mM Standard solution. Mix well.Unused dissolved Standard can be stored in a tightly sealed glass bottle at 4°C protected from light for 1 month.2. Standard Curve SetupDilute the 64 mM Standard further with Extraction Buffer as shown in the table below.Standard No.Standard (µL)Extraction Buffer Volume (µL)Standard Concentration (mM)Std.120µL of 64mM stock6202Std.2100µL of Std.11001Std.3100µL of Std.21000.5Std.4100µL of Std.31000.25Std.5100µL of Std.41000.125Std.6100µL of Std.51000.0625Std.7100µL of Std.61000.0313Note: Prepare freshly diluted standards for each experiment.3. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 6 months.3.1 Animal Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8,000 rpm, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.2 Plant Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and grind. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8,000 rpm, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.3 Cells or Bacteria: Collect 5 million cells or bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8,000 rpm, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.4 Serum (Plasma) and other liquids: Detect directly.4. Assay Steps4.1 Instrument Preparation: Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 550 nm. For spectrophotometers, zero the instrument with deionized water.4.2 Sample Assay (Add reagents sequentially to EP tubes):ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Extraction Buffer240200200Various Std.0400Sample0040Cap the tubes tightly and vortex at medium speed for 30 seconds.Cu Reagent808080Cap the tubes tightly and vortex at medium speed for 30 seconds. Incubate at room temperature (25°C) for 20 minutes. Centrifuge at 2,000 g, room temperature (25°C) for 5 minutes.Upper Phase505050Chromogen2002002004.3 Incubate at room temperature (25°C) for 5 minutes. Transfer 200 µL from each tube to the corresponding wells of a 96-well plate or micro glass cuvettes. Measure the absorbance at 550 nm.Calculate ΔAtest=Atest-Ablank and ΔAstd=Astd-Ablank (The blank tube only needs to be set up once).Note: The measurement must be completed within 30 minutes after color development. It is recommended to perform preliminary experiments with 2-3 samples expected to have significant differences before formal testing. If Atest exceeds the detection range of the instrument, dilute the sample further with Extraction Buffer and multiply the result by the dilution factor.5. Result CalculationWe provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas.5.1 Standard Curve PlottingPlot the standard curve with standard concentration as the y-axis and ΔAstd as the x-axis (using concentration as the y-axis facilitates calculation). Substitute ΔAtest into the standard curve equation to obtain y (mM).5.2 Sample FFA Content Calculation(1) Based on sample mass:FFA Content (µmol/g fresh weight) = y × Vextract ÷ W × n = y ÷ W × n(2) Based on bacterial or cell count:FFA Content (µmol/10⁴ cells) = y ÷ (Cell or Bacterial Count ÷ Vextract ) × n = y ÷ 500 × n = 0.002 × y × n(3) Based on liquid volume:FFA Content (µmol/L) = 1000 × y × nParameter Description:Vextract : Volume of Extraction Buffer added, 1 mLW: Sample mass, gn: Sample dilution factor (if further diluted)500: Cell or bacterial count, in units of 10⁴1000: Unit conversion factor, 1 L = 1000 mL6. Result PresentationTypical Standard Curve: y = 0.679x - 0.0109, R² = 0.9988(Free Fatty Acid (FFA) standard curve analyzed using a 96-well plate. Data and curve are for reference only; users must establish their own standard curve based on their experiment.)Precautions1. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear personal protective equipment such as lab coats, masks, gloves, and hair caps. Perform experiments in a fume hood or biosafety cabinet.2. This product is for scientific research use only. Not intended for clinical diagnosis... Read More | The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, the bacteria with intact cell membrane appear green, while the bacteria with damaged cell membrane can appear green and red under different channels, respectively. A common criterion for bacterial viability is the ability to propagate in a suitable nutrient medium, known as a growth assay. This kit is generally in good agreement with the growth assay results in liquid or solid medium. However, under certain conditions, membrane damaged bacteria may recover and propagate in nutrient medium, and such bacteria will be identified as dead bacteria in this assay. On the contrary, some bacteria with intact membranes may not be able to propagate in nutrient medium, but will be recognized as viable bacteria in this assay. Therefore, if there is a large difference between the test results of this kit and the bacterial growth assay, the above possibilities should be considered. Component： Product parameters： NucGreen: Ex/Em = 503/530 nm (结合 DNA)；EthD-III: Ex/Em = 530/620 nm (结合 DNA)。Usage:1 Preparation of control samples for live and dead bacteria (optional)1. Cultivate 4 mL of bacteria in liquid medium until late logarithmic phase.2. Prepare two 1 mL bacterial solutions in an EP tube and centrifuge for 10-15 minutes under 5000-10000 g conditions.3. Remove the supernatant and add 0.3 mL of 0.85% NaCl resuspended bacteria to one of the EP tubes, and 1 mL of 0.85% NaCl resuspended bacteria to the other tube.4. Add 0.7 mL of isopropanol to a tube containing 0.3 mL of 0.85% NaCl, and mix thoroughly (with a final concentration of 70% isopropanol) to prepare a dead bacterial sample.5. Incubate the two samples at room temperature for 1 hour and mix every 15 minutes.6. Centrifuge the two samples at 5000-10000 g for 10-15 minutes.7. Remove the supernatant, add 1 mL of 0.85% NaCl to resuspend the bacteria in both samples, and centrifuge again as in step 6.8. Use a spectrophotometer to measure the absorbance values (OD670) of two bacterial suspensions at 670 nm.9. Adjust the density of the two bacterial suspensions (live and dead) to 108 bacteria/mL (OD670 ≈ 0.3), and then dilute with 0.85% NaCl at 1:100 to achieve a final density of 106 bacteria/mL.10. Mix two bacterial suspensions as shown in the table below to obtain the required live cell ratio: dead cell ratio.Table 1 Mix live and dead bacterial suspensions by a certain volume to achieve the required ratio of live and dead cellsLive cells: Dead cellsVolume of viable bacterial suspension（mL）Volume of dead bacterial suspension（mL）0:10001.010:900.10.920:800.20.830:700.30.750:500.50.5100:01.00II Staining methods for fluorescence microscopy observation1. Mix 1 volume of component A, NucGreen, and 2 volumes of component B, EthD-III, in a microcentrifuge tube. After thorough mixing, add 8 volumes of 0.85% NaCl solution to obtain a 100 x dye solution.2. Every 100 µ L bacterial suspension, add 1 µ 100 x dye solution of L.3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.4. Take 5 µ The bacterial suspension after L staining was dropped onto a glass slide with an 18 mm square cover glass.5. Observe under a fluorescence microscope. The fluorescence of live and dead bacteria can be observed simultaneously under any standard FITC long-acting filter. Alternatively, live (green fluorescent) and dead (red fluorescent) bacteria can be observed using FITC and Cy3 (or Texas Red) channels, respectively.Attention: (1) Before staining bacteria, attention must be paid to removing residues of growth media. Nucleic acid and other media components can bind to NucGreen and EthD-III dyes in some way, resulting in unacceptable staining changes. A simple washing step is usually sufficient to remove interfering media components from bacterial suspension. It is not recommended to use phosphate buffer solutions as they can reduce staining efficiency. (2) Before starting the formal experiment, the dye concentration should be adjusted to distinguish between NucGreen labeling live bacteria and EthD-III labeling dead bacteria. The optimal concentration may vary depending on the bacterial strain. It is generally best to use the lowest dye concentration that can provide sufficient signal. The above conditions have been optimized for staining live/dead cells of Escherichia coli.III Before starting the staining method experiment of flow cytometry, please read the precautions under the fluorescence microscope staining steps.According to Table 1, add 11 different proportions of live and dead bacteria to the EP tube. Each of the 11 samples has a volume of 1 mL.2. Add 12 µ The A component of L, NucGreen, and 24 µ The B component EthD-III of L was mixed in a microcentrifuge tube. Add 3 to each of the 11 samples µ Mix the mixed dyes of L thoroughly by blowing them up and down several times. (Note: Additional control bacterial samples need to be prepared for separate NucGreen and EthD-III staining)3. Incubate at room temperature in the dark for 15 minutes.4. Analyze each sample using a flow cytometer, detect NucGreen positive cells using FITC channels, and detect EthD-III positive cells using PI or PE channels.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. if the orifice plate is used for detection, a small amount of bacterial liquid can be left for imaging after standing for 10 min, which can effectively reduce the background. 3. in order to be closer to the real results, it is recommended to keep the brightness of red fluorescence consistent with that of green fluorescence in merge pictures. 4. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 5. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Staining of dead and live bacteria... Read More | Product IntroductionBCIP (5-Bromo-4-chloro-3-indolyl phosphate) 5-bromo-4-chloro-3-indolyl-phosphate + NBT (tetrazolium nitro blue) is the best substrate for alkaline phosphatase (AP) One of the combination. Under the catalysis of alkaline phosphatase, BCIP will be hydrolyzed to produce a highly Product IntroductionBCIP (5-Bromo-4-chloro-3-indolyl phosphate) 5-bromo-4-chloro-3-indolyl-phosphate + NBT (tetrazolium nitro blue) is the best substrate for alkaline phosphatase (AP) One of the combination. Under the catalysis of alkaline phosphatase, BCIP will be hydrolyzed to produce a highly reactive product, which reacts with NBT to form an insoluble dark blue to blue-violet compound. This kit can be used for the enzymatic color development of IHC and Western Blot experiments of the AP system. Under AP catalysis, a dark blue precipitate is produced where AP conjugates are combined on tissue sections or blotting membranes. The location and expression of the target protein can be determined based on the color reaction.Product Components40×BCIP: 1 ml40×NBT: 1 mlBCIP/NBT Buffer: 40 mlPrecautions1. The working fluid should be prepared for immediate use, and the prepared working fluid will be effective within 1 hour.2. The amount of working fluid must be sufficient to ensure complete coverage of the tissue sheet or blotting membrane. To3. In order to obtain the best experimental results, be sure to optimize the experimental conditions.4. NBT is poisonous, please take necessary protective measures when using it.5. This product is only used for scientific research, not for human experiments or human treatment.Instructions1. BCIP/NBT color developing working solution preparation:According to the required amount, mix 40×BCIP, 40×NBT and BCIP/NBT Buffer in a volume ratio of 1:1:38 to form the BCIP/NBT color developing working solution.2. Color rendering:1) Blotting membrane color development: Drop the prepared working solution on the blotting membrane (or pour the blotting membrane into the BCIP/NBT color developing working solution), and incubate for 3-10 minutes at room temperature and dark. After the color development is completed, the film is immersed in water to terminate the reaction.2) Color development of tissue sections or cell slides: Drop an appropriate amount of BCIP/NBT color developing working solution on the tissue sections or cell slides that need color development, and incubate at room temperature for 3-10 minutes in the dark. Observe under the microscope to control the color development time. When the best color development effect is reached, rinse with tap water to stop the color development. After color development, the slices are counter-stained, dehydrated and transparent, and can be stored for a long time after mounting... Read More | Inquire | DescriptionWhite LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatableDescriptionWhite LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatable milliamp selection for photon intensity (sold seperately)0-30 mA variable LED outputNon-magnetic LED baseChemically resistant LED coverPTFE coated cablingPhoto Kitalysis Starter Kitrequired for operation (sold separately). Best when used withKitAlysis Benchtop Inertion Box(sold separately)... Read More |