| Description | Lactate dehydrogenase (LDH or LD) is a stable protein present in the cytoplasm of normal cells and normally cannot pass through the cell membrane. When cells are damaged, membrane permeability increases, and LDH is released extracellularly. A decrease in intracellular LDH and an increase in LDH in Lactate dehydrogenase (LDH or LD) is a stable protein present in the cytoplasm of normal cells and normally cannot pass through the cell membrane. When cells are damaged, membrane permeability increases, and LDH is released extracellularly. A decrease in intracellular LDH and an increase in LDH in the culture medium occur. Measuring the LDH activity in the culture medium or the LDH leakage rate can reflect drug-induced cytotoxicity. LDH belongs to the oxidoreductase family and can reversibly catalyze the redox reaction between lactate (L) and pyruvate (P). The reaction formula is: Lactate + NAD⁺ → Pyruvate + NADH + H⁺, where L → P is the forward reaction and P → L is the reverse reaction. Detection Principle: Using NAD⁺ as a hydrogen acceptor, LDH catalyzes the dehydrogenation of lactate to generate pyruvate. Pyruvate then reacts with dinitrophenylhydrazine to form pyruvate dinitrophenylhydrazone, which appears brownish-red in an alkaline solution. The color intensity is proportional to the pyruvate concentration. The absorbance at 440 nm can be measured using a microplate reader. The released LDH activity during cytotoxicity or the LDH activity in other samples can be calculated using formulas. This kit can be used for routine LDH activity detection and is more commonly used for cytotoxicity assays using LDH release as an indicator.This kit is for scientific research use only and is not intended for clinical diagnosis or other purposes.L1501786Component100T500TStorageL1501786ALDH Assay Buffer3 mL15 mL2-8℃. Store in the dark.L1501786BNAD1EA2EA-20℃L1501786CPhenylhydrazine Color Solution3 mL15 mL2-8℃. Store in the dark.L1501786DAlkaline Color Solution10 mL50 mLRT.L1501786ELDH Releasing Agent (10X)2 mL10 mLRT.User-Prepared Instruments and Reagents1. 96-well plate cultured test and control group cell samples, sterile PBS, culture medium, distilled water.2. Microplate centrifuge, 96-well plate or centrifuge, centrifuge tubes, incubator or water bath, microplate reader.Experimental Procedure1. Sample Preparation1.1 LDH Release AssaySeed an appropriate number of cells into a 96-well culture plate based on cell size and growth rate, so that the cell density does not exceed 90% confluency at the time of detection.Aspirate the culture medium, wash once with PBS, add fresh culture medium.Set up corresponding control groups according to experimental needs:Background Blank Control Well A: Culture medium without cells.Sample Control Well B: Control cells without drug treatment.Maximum Enzyme Activity Control Well C: Lysed samples from untreated cells.Drug-treated Sample Well D: Cells treated with the drug.Continue cultivation.Before detection, take out the cell culture plate. Add LDH Releasing Agent (10X) to the "Maximum Enzyme Activity Control Well C" at a volume equal to 10% of the original culture medium volume. Mix thoroughly by pipetting up and down several times. Continue cultivation for about 1 hour.Centrifuge the cell culture plate at 400 g for 5 minutes using a microplate centrifuge.Aspirate 5 µL of supernatant from each well and transfer it to the corresponding wells of a new 96-well plate for subsequent LDH detection.1.2 Cytotoxicity and Cell Proliferation Assay for Intracellular Total LDHSeed an appropriate number of cells into a 96-well culture plate based on cell size and growth rate, so that the cell density does not exceed 90% confluency at the time of detection.Treat with different drugs and set up appropriate controls.Centrifuge the cell culture plate at 400 g for 5 minutes using a microplate centrifuge.Aspirate the culture medium.Add 150 µL of LDH Releasing Agent diluted 10-fold with PBS. Shake the plate to mix thoroughly. Continue cultivation for about 1 hour.Centrifuge the cell culture plate at 400 g for 5 minutes using a microplate centrifuge.Aspirate 5 µL of supernatant from each well and transfer it to the corresponding wells of a new 96-well plate for subsequent cytotoxicity detection.1.3 Protein Concentration DeterminationAfter sample preparation, the protein concentration can be determined using a BCA Protein Assay Kit (Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit are recommended) to facilitate subsequent calculation of LDH content per unit protein weight in tissues or cells.2. Preparation of NAD SolutionTake one vial of NAD (powder) and dissolve it in 1.5 mL of deionized water.3. LDH Enzymatic ReactionAdd solutions sequentially according to the table below, taking care to avoid bubbles. If the enzyme activity in the sample is too high, reduce the sample volume or dilute appropriately before assay.Reagent (µL)Volume (µL)Test Sample (supernatant)5LDH Assay Buffer25NAD Solution5 Mix well, incubate at 37°C for 15 min. Phenylhydrazine Color Solution25 Mix well, incubate at 37°C for 15 min. Alkaline Color Solution100Distilled Water150 4. LDH Measurement Mix well and let stand at room temperature for 5 minutes. Measure the absorbance of each well at 440 nm using a microplate reader. 5. Result Calculation Cytotoxicity or Mortality Rate (%) = (A D - A B ) / (A C - A B ) × 100% If the absorbance value A γ of a known concentration *c* of an LDH enzyme standard and the absorbance value A γ0 of the standard blank control are measured simultaneously, the enzyme activity in the sample can be roughly calculated:LDH Activity in Test Sample (mU/mL) = (A B - A A ) / (A γ - A γ0 ) × *c* For accurate calculation of the absolute LDH enzyme activity in the sample, use a self-prepared LDH standard to plot a standard curve with the measured absorbance values. The enzyme activity of the sample can be calculated using the formula derived from the standard curve. Where: A A = Absorbance of Background Blank Control Well A A B = Absorbance of Sample Control Well B A C = Absorbance of Maximum Enzyme Activity Control Well C A D = Absorbance of Drug-treated Sample Well D 6. Results and Analysis The cytotoxicity of drugs or toxicants can be determined by directly comparing the LDH activity in each well. Higher LDH activity indicates higher cell membrane permeability and more severe cell damage.Precautions1. Use serum-free or low-serum concentration culture medium when culturing cells to exclude serum interference; otherwise, deviations may occur.2. EDTA inhibits LDH. Avoid using or thoroughly remove reagents containing EDTA during operation.3. Measure LDH as soon as possible after collection. If the collected cell culture medium is stored for too long, LDH activity may decrease.4. Use solutions prepared at the same time for the same batch of experiments. The volume of solutions used and the reaction time should be consistent.5. In the enzymatic reaction, the recommended supernatant sample volume is 2.5-10 µL. If the enzyme activity in the sample is too high, reduce the sample volume or dilute appropriately before assay.6. Measurement should be completed within 15 minutes after color development.7. The Alkaline Color Solution is somewhat corrosive; handle with care.8. Use reagents promptly after opening to avoid affecting subsequent experimental results.9. For your safety and health, please wear a lab coat and disposable gloves during operation... Read More | CFDASE cell proliferation and tracking detection kit is a kit for cell proliferation and tracking detection based on CFDA se. This kit is composed of CFDASE powder, solvent and staining buffer. CFDASE is a derivative of fluorescein diacetate (FDA), which has cell membrane permeability and CFDASE cell proliferation and tracking detection kit is a kit for cell proliferation and tracking detection based on CFDA se. This kit is composed of CFDASE powder, solvent and staining buffer. CFDASE is a derivative of fluorescein diacetate (FDA), which has cell membrane permeability and does not have fluorescence luminescence. When CFDASE penetrates the cell membrane into living cells, it can be catalysed by esterases in the cytosol to produce carboxyfluorescein succinimidyl ester (CFSE), which can emit strong green fluorescence, cannot penetrate the cell membrane, and can remain intact in the cell. CFSE can also spontaneously and irreversibly covalently bind to intracellular amino groups to couple to cellular proteins. Meanwhile, the excess and uncoupled CFDASE returned to the extracellular medium by passive diffusion and was cleared by subsequent washing steps. The fluorescence of non dividing cells labeled by CFDASE is very stable, and the stable labeling time can reach several months, so it is very suitable for cell community analysis. The fluorescence of CFDASE labeled cells is very homogeneous, which is superior to other cell tracking fluorescent probes used previously, such as PKH26, and the fluorescence distribution of the divided progeny cells is also very uniform. In the process of cell division and proliferation, CFSE labeled fluorescence can be evenly distributed to the two progeny cells, and the fluorescence intensity becomes half of the parental cells. According to the fluorescence intensity, flow cytometer (FL1 channel) can detect undivided cells, cells that divide once (1 / 2 of the fluorescence intensity), twice (1 / 4 of the fluorescence intensity), three times (1 / 8 of the fluorescence intensity), and cells that divide more times. CFDASE can detect up to eight or more cleavages. CFDASE labeled cells can be used for proliferation studies in vitro and in vivo, and have the function of not staining adjacent cells. CFDASE is most commonly used to detect the proliferation of lymphocytes, and can also be used to detect the proliferation of fibroblasts, NK cells and other cells. CFDASE labeled cells showed green fluorescence. In addition to flow cytometry to detect cell proliferation, fluorescence microscopy can also be used for homogeneous staining of cell tracking observation.Components:ComponentsC598182-20TC598182-500TA. CFDA SE1 tube1 tubex5B.CFDA SE solvent20 µL500 µLC.10x CFDA SE Buffer1 mL x250 mLMatters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. CFDA and Se are easily hydrolyzed and will deteriorate quickly in aqueous solution. Please avoid contact with water during use. Contact with water during the process of labeling cells is within the permitted range. 3. CFDA se solvent will solidify at lower temperatures such as 4 º C and ice bath and stick to the bottom, wall or cover of the centrifugal tube. It can be used after incubating in a 20-25 º C water bath for a while until it is completely dissolved. 4. this kit optimizes the CFDA se staining system, but users are advised to explore the optimal working concentration and staining time according to their own cell type, culture conditions and application direction. Different cells have different lactonase activities, so the staining effect is different. 5. fluorescent dyes have quenching problems. Please avoid light during operation to slow down fluorescence quenching. 6. for your safety and health, please wear experimental clothes and disposable gloves.Usage method:1. Preparation of reagents(1) Preparation of CFDA SE storage solution: Take one tube of CFDA SE provided in the reagent kit and restore it to room temperature. Instantly centrifuge to allow the powder to fully settle to the bottom of the tube. Add 100 µ L CFDA SE solvent (add 20 µ L CFDA SE solvent) to it and dissolve it thoroughly to prepare CFDA SE storage solution (1000 ×). Prepared CFDA SE storage solution, stored at -20 ℃ in the dark, with a shelf life of two months- Storing at 70 ℃ in the dark can extend the usage time appropriately.(2) Preparation of CFDA SE Buffer: Dilute 10 x CFDA SE Buffer to 1 x with sterile cell culture grade water as needed. The prepared 1 × CFDA SE Buffer can be stored at 4 ℃ and can be stored at -20 ℃ if not in use for a long time.2. Marking and detection(1) Centrifuge the collected cells, use 1 mL 1 × CFDA SE Buffer to re suspend the cells in a 15 mL centrifuge tube, and adjust the cell concentration to 1-5 × 106 cells/mL.(2) Preparation of CFDA SE working solution: Dilute the CFDA SE storage solution (1000 ×) with 1 × CFDA SE Buffer to 2 ×.(3) Staining: Add 1 mL of CFDA SE working solution (2 x) to 1 mL of cell suspension to be labeled, invert and mix well, and incubate at 37 ℃ for 10 minutes.(4) Immediately add 5 times the volume of preheated complete culture medium (including serum) to the centrifuge tube, invert and mix well to terminate the labeling reaction.(5) Centrifuge at 1000 rpm for 5 minutes at room temperature to remove the supernatant, then wash once with 5-10 mL of complete culture medium.(6) Add 5-10 mL of complete culture medium and incubate at 37 ℃ for 5 minutes to promote the residence of CFDA SE in the cells and the entry of unreacted CFDA SE into the complete cell culture medium. Centrifuge at 1000 rpm for 5 minutes at room temperature to remove the supernatant and complete the final wash.(7) Subsequently, the cells can be cultured using the normal cultivation method. The labeling effect can be directly observed under a fluorescence microscope, or cell proliferation can be detected by flow cytometry after appropriate cultivation time, showing green fluorescence. The labeled cells can also be used for transplantation in live animals and for fluorescence tracing.Note: a If cell fixation is required, use aldehyde fixatives such as 4% paraformaldehyde to fix at room temperature for 15 minutes; If additional labeling such as antibody labeling is required afterwards, please permeabilize the cells with ice acetone for 10 minutes. b. The optimal labeling concentration and incubation time for CFDA SE vary for different cells. The initial experiment can be conducted according to the experimental steps. If the effect is not satisfactory, it is recommended to adjust the staining concentration and incubation time to achieve the best labeling effect.Scope of application：Cell proliferation assay... Read More | DescriptionThe 1 µm Coupling Kit makes conducting immunoprecipitation and biomolecule separation easier and more flexible. The Kit contains AnteoBind™activated 1 µm magnetic particles that give you increased antibody binding capacity and functionality, while the included blocking DescriptionThe 1 µm Coupling Kit makes conducting immunoprecipitation and biomolecule separation easier and more flexible. The Kit contains AnteoBind™activated 1 µm magnetic particles that give you increased antibody binding capacity and functionality, while the included blocking buffer decreases background noise. Reduce reagent preparation time; remove traditional surface preparation steps such as EDC and replace these steps with the 1 µm pre-activated magnetic particles provided. This Kit reduces aggregation and gives you the freedom and ability to develop multifunctional particles for diverse applications, including dual labelling.Binding Capacity and Dispersity:Binding Capacity:> 20 µg IgG/mgMonodispersity:> 90% (by light microscopy determination)Particle based immunoassays, bioseparations and immunoprecipitation... Read More | Product introduction： The MA qPCR live bacteria detection kit provides an effective means for detecting bacterial activity. The kit provides a mixture of PMA dye and qPCR based on SYBR Green dye. The optimal amount of dye and the number of samples that can be treated may vary depending on theProduct introduction： The MA qPCR live bacteria detection kit provides an effective means for detecting bacterial activity. The kit provides a mixture of PMA dye and qPCR based on SYBR Green dye. The optimal amount of dye and the number of samples that can be treated may vary depending on the type of sample. PMA is a high-affinity DNA-binding dye, especially with double-stranded DNA. The dye itself has weak fluorescence, but it can emit brighter fluorescence after binding to nucleic acids. PMA is impermeable to cell membranes, so it can selectively modify the DNA of dead cells with damaged membranes. After the PMA-modified DNA is photolyzed by blue light ( ~ 464 nm ), the photoreactive azide group on the PMA is converted into a highly reactive nitrene radical, which reacts with any hydrocarbon near the DNA binding site to form a stable covalent nitrogen-carbon bond, resulting in permanent DNA modification. This modification process will make DNA insoluble and lost with cell debris during the later genomic DNA extraction process. The unbound PMA remaining in the solution reacts with water molecules under strong light irradiation to decompose into hydroxylamine compounds without cross-linking activity, so that it can no longer covalently bind to DNA. Based on this feature of PMA, PMA was combined with qPCR technology to form a new detection method, PMA-qPCR, for the screening of live bacteria. At present, the method has been verified in a variety of bacterial strains, yeast, fungi, viruses and parasites. The treatment of complex samples, such as manure or soil, may require optimization of sample dilution, dye concentration, and light treatment time. The treatment of diluted samples, such as water testing, may require filtration or concentration prior to dye treatment. Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. the components of the kit contain fluorescent dyes. Avoid light during use and storage. 3. for your safety and health, please wear experimental clothes and disposable gloves.Product parameters:Spectral characteristics :PMA: Ex = 464 nm; Ex/Em = 510/610 nm (following photolysis and reaction with DNA/RNA)Component： PMA：Ex = 464 nm; Ex/Em = 510/610 nm (following photolysis and reaction with DNA/RNA) Instruction： Precautions before use： 1.This live bacteria detection kit distinguishes dead bacteria and live bacteria according to cell membrane permeability. Many methods of killing bacteria cause damage to the cell membrane and are therefore compatible with this kit. But some methods, such as ultraviolet irradiation, may not immediately cause cell membrane rupture. Therefore, before selecting this kit, it is necessary to carry out literature search and pre-experiment to determine whether the kit is suitable for the bacterial type and killing method you choose. 2.After PMA treatment, the bacteria need to be photolyzed to covalently bind the dye to dead cell DNA. Photolysis operations can use blue or white light sources. Generally speaking, the brighter the lamp, the higher the efficiency of the photolysis step. Non-LED lamps ( such as halogen lamps ) may heat your sample and have a negative impact on the analysis. Ice is required to cool the sample during irradiation. 3.Sample can be cryopreservation after photolysis. Frozen samples before PMA treatment photolysis may damage the cell membrane and produce false negative results. If the sample needs to be frozen before detection, it is recommended to perform a pre-experiment first. 4.Part of the mechanism of PMA is to remove PMA covalently modified DNA from the sample by precipitation ; therefore, when extracting genomic DNA, it is necessary to use the same volume of genomic DNA eluent for volume normalization. The positive control can use the genomic DNA of living cells. 5.In order to verify the effectiveness of PMA in the test sample, the Ct ( dCt ) changes between- / + PMA can be compared. Experimental materials ( self-provided )：①Light source ( for the photolysis step after PMA modification of DNA ) ; ② Bacterial genomic DNA extraction kit ; ③ effective qPCR primers corresponding to the sample type Experimental procedure： 1.Suck 10 µL of E.coli bacterial solution in liquid LB medium, and culture E.coli in the bacterial incubator overnight or longer to the logarithmic growth phase ( OD600 ≈ 1.0 ) ; Note : The culture time is adjusted according to the experiment. 2.Two portions of live E.coli, 400 µL each, were placed in a clean centrifuge tube ; 3. ( Recommended ) Preparation of dead E.coli. If the dead E.coli is needed as a control, the dead E.coli can be obtained by heating the living E.coli in a water bath at 95 °C for 5 min, or at 58 °C for 3 h. the subsequent operation of the dead E. coli is the same as that of the living E. coli ; 4.Two copies of live E.coli, one without PMA treatment, and one with 25 µM PMA treatment ( the optimal PMA concentration for treating different types or different sources of bacteria needs to be consulted in the relevant literature ) ; 5.The PMA-treated samples were placed on a shaker at room temperature and incubated in the dark for 10 min to fully mix the dye with the sample ; 6.Exposure of the sample, you can use blue or white light source, irradiation time to explore their own. For example, a 60 W blue light can be used for 15 min. Note : 1 If a halogen lamp is used, we recommend that the PMA-treated sample tube be placed on an ice block 20 cm away from the light source. Ice should be placed in a transparent tray. Adjust the light source to point directly to the sample, photolysis for 5-15 min ; if the bacteria obtained from the environment are directly used for experiments, due to the complexity or turbidity of the environmental samples, the photolysis time needs to be prolonged appropriately. 7.Treated and untreated live E.coli 5000 × g, centrifuged for 10 min, remove the supernatant ; 8.Select the appropriate genomic DNA extraction kit according to the sample type, and use the same elution volume for each group of samples when elution DNA. Note : DNA extraction steps refer to the instructions of the kit used. Part of the mechanism of action of PMA is to remove PMA-bound DNA from the sample by precipitation ; therefore, when extracting genomic DNA, each group should use the same volume of genomic DNA eluent for volume normalization ( the amount of genomic DNA extracted from dead bacteria and live bacteria is inconsistent, so the concentration of the two is significantly different ). 9.Preparation of reaction mixture according to the following system : Note : 1 For the DNA extracted by commercial DNA extraction kit, the qPCR template was optimized with 2 µL as the initial volume ; 2 The template volume should not exceed 10 % of the final reaction volume ; 3 Template concentration : gDNA as template, usually 1-10 ng ; the final concentration of PCR primers is usually 0.4µM, which can get better results. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.2-1µM. 10.Slightly vortex the reaction mixture, transfer the fixed volume to the PCR tube. 11. Test procedure Note : 1 The extension time is adjusted according to the instrument ; the Taq enzyme in mix can be activated within 2 min, but the genomic DNA may require longer denaturation time, which can be increased at this time, and the specific denaturation time can be adjusted according to the sample type.12. ( Optional ) Data analysis Using live bacteria and dead bacteria as controls, the number of live cells in the sample was analyzed and calculated. It is recommended to verify the suitability of primers and PCR procedures before starting PMA qPCR detection of live bacteria. Calculation of dead and living bacteria control dCt ( 1 ) After the end of qPCR, the Ct value of each sample was calculated by instrument software ; ( 2 ) By calculating the dCt of each control bacteria, it was judged whether PMA successfully inhibited the amplification of dead bacterial DNA. The calculation is as follows : dCt live = Ct ( live, PMA treated ) -Ct ( live, PMA untreated ) dCt die = Ct ( die, PMA treated ) -Ct ( die, PMA untreated ) ( 3 ) The dCt expectation of living bacteria is close to 0 ± 1, which indicates that PMA does not affect the amplification of living cell DNA ;( 4 ) The expected value of dCt of dead bacteria is greater than 4 ( dCt is 4 means that it is reduced by about 16 times, that is, 94 % of dead bacterial DNA is removed ; a dCt of 8 indicated a decrease of about 250 times, that is, 99.6 % of the dead bacterial DNA was removed ).( 5 ) The dCt of dead bacteria depends on many factors, including : strain / cell type ; the way bacteria are killed ; the concentration of PMA used ; amplified sequence length. 13. Calculation of the proportion of viable ( optional ) bacteria If the control results of dead and live bacteria are normal, the proportion of live bacteria in the sample can be calculated.( 1 ) Calculate the dCt value of the sample : dCt sample = Ct ( sample, PMA treated ) -Ct ( sample, PMA untreated ) ( 2 ) Conversion of dCt value to live bacteria ratio : PMA inhibition multiple = 2 ( sample dCt ) Viable bacteria % = 100 / PMA inhibition multiple 14. ( Optional ) Calculate the absolute number of live bacteria If you want to calculate the absolute number of viable bacteria in the sample, you need to use a known number of target bacteria genomic DNA to make a standard curve. It is recommended that the diluted concentrations of several groups of genomes are within the range of the qPCR analysis system.( 1 ) qPCR was performed with the appropriate genome, and the Ct value was used as the ordinate, and the number of cells was used as the abscissa. The R2 value is calculated to determine the linearity, and the slope and y-axis intercept are displayed. ( 2 ) Calculate the copy number of the experimental samples : Ct = slope * cell number + y axis intercept ( y = mx + b ) Bacterial count sample = ( Ct-y axis intercept ) / slope Note : The live bacterial DNA was not lost during the purification process. Examples : Scope of application：Live bacteria detection... Read More | Inquire |