| Description | Low-Density Lipoprotein (LDL) in plasma is the main carrier of endogenous cholesterol. It is degraded and metabolized by binding to the LDL receptor (LDL-R) on cell membranes and serves as the primary vehicle for transporting cholesterol to peripheral tissues. However, when LDL, especially oxidized Low-Density Lipoprotein (LDL) in plasma is the main carrier of endogenous cholesterol. It is degraded and metabolized by binding to the LDL receptor (LDL-R) on cell membranes and serves as the primary vehicle for transporting cholesterol to peripheral tissues. However, when LDL, especially oxidized LDL (OX-LDL), is present in excess, the cholesterol it carries accumulates in the arterial walls, increasing the risk of atherosclerosis. Atherosclerosis is the pathological basis and risk factor for the majority of cardiovascular and cerebrovascular diseases.Detection Principle: In a cholesterol assay system containing cholesterol esterase (CHER) and cholesterol oxidase (CHOD), specific surfactants are added to selectively solubilize LDL-C for the determination of LDL-cholesterol. Other lipoproteins (HDL, VLDL, chylomicrons) do not react due to inhibition by the surfactants and sugar compounds, remaining in the form of lipoproteins in the reaction mixture. Based on this principle, LDL-cholesterol can be measured directly. Subsequently, esterase catalyzes the hydrolysis of cholesterol esters to generate Free Cholesterol (FC). FC is oxidized by cholesterol oxidase to produce 4-cholestenone and hydrogen peroxide. Hydrogen peroxide then reacts with 4-aminoantipyrine and other components to produce a red quinoneimine compound, which has a characteristic absorption peak at 546 nm. The LDL-C content is determined by measuring the absorbance at 546 nm.Component96TStorageReagent 118 mL2-8℃. Store in the dark.Reagent 26 mL2-8℃. Store in the dark.Standard1EA2-8℃. Store in the dark.Standard (Powder, 1 vial) Preparation:1. Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom of the tube.2. Add 0.1 mL of distilled water to dissolve. Use within one week. The prepared concentration is as indicated on the label.User-Prepared Instruments and Reagents:Mortar (Homogenizer), balance, ice box (ice maker), benchtop centrifuge, adjustable micropipettes, water bath (oven, incubator, metal bath), 96-well plate, centrifuge tubes, microplate reader, distilled water (deionized water or ultrapure water are acceptable), ethanol.Experimental ProcedureIt is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.1. Sample Extraction1.1 Tissue SamplesWeigh approximately 0.1 g of tissue sample and place it in a mortar. Add 1 mL of ethanol and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C or room temperature for 10 minutes. Collect the supernatant for assay.Note: If increasing the sample amount, maintain a tissue mass (g) to ethanol volume (mL) ratio between 1:5 and 1:10.1.2 Liquid SamplesAssay clear liquid samples directly. If turbid, centrifuge and use the supernatant for assay.1.3 Bacterial/Cell SamplesCollect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of ethanol per approximately 5 million bacteria/cells. Disrupt the bacteria or cells by sonication in an ice bath (power 200W, pulse 3s on, 10s off, repeat 30 times). Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.*Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to ethanol volume (mL) ratio between 500:1 and 1000:1.*2. Assay Steps2.1 Preheat the microplate reader for 30 minutes (or wait for the instrument to complete its self-check). Set the wavelength to 546 nm.2.2 Thaw all reagents to room temperature (25°C). Add reagents sequentially to a 96-well plate as follows:Reagent (µL)Test TubeStandard Tube (once)Blank Tube (once)Sample2.5Standard2.5Distilled Water2.5Reagent 1180180180Mix well and incubate at 37°C for 5 minutes. Read the absorbance at 546 nm for each tube (A1 ).Reagent 2606060Mix well and incubate at 37°C for 10 minutes. Read the absorbance at 546 nm for each tube (A2 ). Calculate ΔA = A2 - A1 for each tube.Note:(1) If the A2 value for the Test Tube is greater than 1, dilute the sample with ethanol. The dilution factor (D) must be substituted into the calculation formula.(2) If ΔA for the Test Tube is lower than ΔA for the Blank Tube, consider increasing the sample volume V1 (e.g., increase the sample volume in the Test Tube and the water volume in the Blank Tube to 5 µL or more, keeping Reagents 1 and 2 volumes unchanged; for the Standard Tube, keep at 2.5 µL and add 2.5 µL distilled water to make up volume) or increasing the sample weight W (e.g., to 0.2 g or more). The changed V1 or W must then be substituted into the calculation formula.3. Calculation of Results3.1 Based on Sample MassDerived Formula:LDL-C (µmol/g weight) = (CStandard × V2 ) × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ (W × V1 ÷ V) × DSimplified Formula:LDL-C (µmol/g weight) = CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ W × D3.2 Based on Protein ContentDerived Formula:LDL-C (µmol/mg prot) = (CStandard × V2 ) × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ (Cpr × V1 ÷ V) × DSimplified Formula:LDL-C (µmol/mg prot) = CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ Cpr × D3.3 LDL-C Content in LiquidsDerived Formula:LDL-C (mmol/L) = (CStandard × V2 ) × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ V1 × DSimplified Formula:LDL-C (mmol/L) = CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) × D3.4 Based on Cell CountDerived Formula:LDL-C (nmol/10⁴ cells) = (CStandard × V2 ) × 10³ × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ (500 × V1 ÷ V) × DSimplified Formula:LDL-C (nmol/10⁴ cells) = 2 × CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) × DParameter Definitions:CStandard : Concentration as indicated on the label (mmol/L or µmol/mL)V1 : Volume of sample added (0.0025 mL)V: Volume of extraction buffer (ethanol) added (1 mL)V2 : Volume of standard added (0.0025 mL)D: Dilution factor (1 if not diluted)500: Number of cells (in units of 10⁴)W: Sample weight (g)Cpr: Protein concentration of the supernatant (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.Precautions1. It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure. Based on the preliminary results, determine or adjust sample concentrations to prevent unnecessary waste of samples or reagents.2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation... Read More | Calcein AM /PI Double Staining Kitis utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stains viable and dead cells, respectively(Fig. 1). Calcein-AM, an acetoxymethyl ester of calcein, is highly Calcein AM /PI Double Staining Kitis utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stains viable and dead cells, respectively(Fig. 1). Calcein-AM, an acetoxymethyl ester of calcein, is highly lipophilic and cell membrane permeable. Though Calcein-AM itself is not a fluorescent molecule, the calcein generated from Calcein-AM by esterase in a viable cell emits a strong green fluorescence (excitationat 490 nm, emission at515 nm). Therefore, Calcein-AM only stains viable cells. On the other hand, PI, a nuclei staining dye, cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered areas of dead cell membrane, and intercalates with the DNA double helix of the cell to emit red fluorescence (excitation: 535 nm,emmision: 617 nm). Since both calcein and PI-DNA can be excited with 490 nm, simultaneous monitoring of viable and dead cells is possible with a fluorescence microscope. With 545 nm excitation, only dead cells can be observed (Fig. 1). Since optimal staining conditions differ from cell line to cell line, we recommend that a suitable concentration of PI and Calcein-AM be individually determined. Please note that PI is suspected to be highly carcinogenic;careful handling is required.Required Equipment and Materials:Microscope with 490 nm excitation filter and 530 nm emission filter；CO2incubator；10 µl and 200 µl adjustable pipettes, PBSSolution A (Calcein-AM)；Solution B (PI) Storage Condition: -20oC ；Shipping Condition: blue ice.Application:Assay Procedure1)Add 2.5 µl Solution A and 12.5 µl Solution B to 5 ml PBS to prepare assay solution.*2)Wash the cell with PBS several times to remove residual esterase activity.3)Add 100uLof assay solution to200uL105~106CELLSsolution and incubate the mixture at 37oC for 15 min.4)Detect fluorescence using a fluorescence mircoscope with 490 nm excitationfor simultaneous monitoring of viable and dead cells.With 545 nm excitation, only dead cells can be observed.*The following steps may be necessary tooptimizethe suitable concentration of each reagent:1)Prepare dead cells by 10 min incubation in 0.1% saponin or 0.1-0.5% digitonin or by 30 min incubation in 70% ethanol.2)Stain dead cells with 0.1-10 µM PI solution to find a PI concentration that stains the nucleus only, not the cytosol.3)Stain dead cells with 0.1-10 µM Calcein-AM solution to find a Calcein-AM concentration that does not stain the cytosol. Then stainviable cells with that Calcein-AM solution to check whether the viable cell can be stained... Read More | H665581 Component 100 T Storage H665581A gDNA Eraser 50 µL -20℃. Avoid freeze/thaw cycle. H665581B 10×gDNA Eraser Buffer 120 µL -20℃. Avoid freeze/thaw cycle. H665581C HiFiScript, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665581D 5×ScriptRT H665581 Component 100 T Storage H665581A gDNA Eraser 50 µL -20℃. Avoid freeze/thaw cycle. H665581B 10×gDNA Eraser Buffer 120 µL -20℃. Avoid freeze/thaw cycle. H665581C HiFiScript, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665581D 5×ScriptRT Buffer 500 µL -20℃. Avoid freeze/thaw cycle. H665581E Primer Mix 120 µL -20℃. Avoid freeze/thaw cycle. H665581F RNase-Free Water 2×1 mL -20℃. Avoid freeze/thaw cycle.Product IntroductionThis product is a kit for removing genomic DNA for reverse transcription. The kit removes genomic DNA in 2 minutes at 42°C. Since the reverse transcription reagent contains a component that inhibits gDNA Eraser, cDNA can be synthesized directly by reverse transcription of gDNA Eraser-treated samples.The kit is equipped with a new high-efficiency reverse transcription enzyme, HiFiScript, with novel mutation sites that dramatically increase the transcriptional activity of the enzyme, resulting in higher efficiency and yield of cDNA first-strand synthesis. The first strand of cDNA can be synthesized with higher efficiency and yield, and the first strand of cDNA can be synthesized from pg total RNA or mRNA. If the reverse transcription product cDNA is used for downstream fluorescence quantitative detection, the reverse transcription reaction can be completed at 42℃ in 15 minutes. This kit is suitable for the synthesis of first-strand cDNA and subsequent RT-PCR, RT-qPCR, and the construction of full-length cDNA libraries.Product Features1. Rapid genome removal: contains gDNA Eraser for genomic DNA removal, which removes genomic DNA in just 2 minutes.2. Rapid reverse transcription: 15 minutes to obtain fluorescent quantitative PCR template cDNA first strand synthesis.3. High sensitivity: cDNA first strand can be synthesized using pg-level total RNA or mRNA templates.4. Highly efficient reverse transcription: Novel mutation sites dramatically increase enzyme activity, resulting in higher yields of cDNA.matters needing attention1. During operation, RNase contamination should be avoided to prevent RNA degradation or cross-contamination in the experiment. It is recommended that operators wear masks and disposable gloves and change the gloves frequently, and use specialized instruments and consumables.2. The reverse transcription system is prepared and operated on ice to prevent degradation of RNA. Store the kit enzymes at -20ºC as soon as possible after use and try to avoid repeated freezing and thawing.3. The reaction system can be scaled up to a maximum of 1 µg of total RNA in 10 µl of reaction system.4. Primer Mix is prepared by Oligo(dT) and Random primer, and Oligo-dT Primer or Gene Specific Primer can also be used according to the experimental needs.5. If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).6. For RNA templates with complex secondary structures, it is recommended to incubate the template RNA at 65°C for 5 minutes immediately on ice prior to the manipulation step and centrifuge briefly before proceeding to the next step.UsageThaw template RNA on ice; place kit components on ice immediately after thawing at room temperature. Each solution was mixed by vortexing and shaking before use and briefly centrifuged.I. Genomic DNA removal reactions1. Prepare the reaction system according to the following table on ice in a total volume of 10 µl. To ensure the accuracy of the reaction solution preparation, prepare the premixed system in the amount of reaction number + 2 before dispensing it into each reaction tube and finally adding the RNA sample.Note: 1) If the amount of total RNA is greater than 1µg, scale up the reaction system proportionally. If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).2. Mix by vortex shaking and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.3. Incubate at 42°C for 2 minutes (this can be extended to 30 minutes for room temperature reactions).4．At the end of the reaction, centrifuge briefly and place on ice to cool.II. Reverse transcription reaction1. Prepare the reaction system on ice according to the following table. In order to ensure the accuracy of the reaction solution configuration, first prepare a premixed solution in the amount of number + 2, and then dispense 10 µl into each reaction tube, take 10 µl of the prepared premixed solution and add it to the reaction tube of step 1 where the de-etching of the genome has been completed.Note: 1) Oligo-dT Primer or Gene Specific Primer can be used according to the needs of the experiment, it is recommended to use 50 pmol of Oligo-dT Primer or 2 pmol of Gene Specific Primer for 20 µl reaction system.2. Mix well and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.3. cDNA synthesis reaction conditions:1) If fluorescent quantitative PCR assay is performed downstream, incubate at 42°C for 15 minutes and 85°C for 5 minutes.2) If downstream for normal PCR assay, incubate at 42°C for 30-50 minutes and 85°C for 5 minutes. Note: For templates with complex secondary structure or high GC content, the reverse transcription temperature can be increased to 50°C to enhance reverse transcription efficiency.4. At the end of the reaction, centrifuge briefly and place on ice before proceeding with subsequent PCR or fluorescence quantitative PCR, or place at -20°C if prolonged storage is required.Note: When performing Real-time PCR reactions, the amount of reverse transcription product added should not exceed 1/10 of the total volume of the PCR reaction... Read More | Inquire | Inquire |