| Description | IntroductionHexokinase (HK) is widely present in animals, plants, microorganisms, and cultured cells. It is the first key enzyme in the glucose degradation pathway, catalyzing the conversion of glucose to glucose-6-phosphate, which is the intersection point of glycolysis and the pentose phosphate IntroductionHexokinase (HK) is widely present in animals, plants, microorganisms, and cultured cells. It is the first key enzyme in the glucose degradation pathway, catalyzing the conversion of glucose to glucose-6-phosphate, which is the intersection point of glycolysis and the pentose phosphate pathway.Assay PrincipleHK catalyzes the synthesis of Glucose-6-Phosphate (G6P) from Glucose. Glucose-6-Phosphate Dehydrogenase (G6PDH) then further catalyzes the dehydrogenation of G6P, generating NADPH. NADPH has a characteristic absorption peak at 340 nm.Component50TStorageExtraction Buffer60 mL2-8℃Reagent 130 mL2-8℃Reagent 21EA2-8℃Reagent 35 mL2-8℃Reagent 41EA-20℃Reagent 51EA-20℃Reagent 61EA-20℃Reagent 2: Powder × 1 bottle. Dissolve in 30 mL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.Reagent 4: Powder × 1 tube. Dissolve in 4 mL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.Reagent 5: Powder × 1 tube. Dissolve in 2 mL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.Reagent 6: Powder × 1 tube. Dissolve in 250 µL Reagent 1 and 250 µL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.Required Materials and Equipment (Not Provided)UV spectrophotometer, constant temperature water bath, benchtop centrifuge, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.Sample Preparation:Bacteria or Cultured Cells:Collect cells by centrifugation and discard the supernatant.Add Extraction Buffer at a ratio of 1 ml per 5-10 million cells (e.g., 1 ml for 5 million cells).Sonicate on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times).Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.Tissues:Homogenize tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., 0.1 g tissue in 1 ml buffer).Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.Serum (or Plasma) Samples:Assay directly.Assay Procedure:Preheat the spectrophotometer for at least 30 min. Set wavelength to 340 nm. Zero with distilled water.Pre-warm Reagents 1, 2, 3, 4, and 5 to 37°C (for mammalian samples) or 25°C (for other species) for 10 min.Pipette into a 1 ml quartz cuvette in the following order:ReagentVolume (µL)Reagent 1400Reagent 2400Reagent 380Reagent 480Reagent 540Reagent 68Sample30Mix immediately upon sample addition and start the timer.Record the initial absorbance (A₁) at 20 seconds and the final absorbance (A₂) at 5 minutes and 20 seconds (320 sec total) at 340 nm.Calculate ΔA = A₂ - A₁.Notes:To minimize operational error, it is recommended to pre-mix Reagents 1, 2, 3, 4, and 5 in the stated proportions. Pre-warm this Master Mix for 10 min. Then add 30 µl sample + 8 µl Reagent 6 + 1 ml Master Mix to the cuvette. Mix and proceed with the assay.HK activity varies across different tissues. Perform a pilot test with 1-2 samples before formal assay. If ΔA > 0.5, the tissue activity is too high. Dilute the supernatant with Extraction Buffer (include dilution factor D in calculations) or shorten the reaction time to 2 min to ensure ΔA < 0.5 and improve detection sensitivity.HK Activity Calculation:General Parameters:Vₜₒₜₐₗ (Total reaction volume) = 1.038 × 10⁻³ L (1038 µL)ε (NADPH molar extinction coefficient) = 6.22 × 10³ L/mol/cmd (Cuvette light path) = 1.0 cmVₛₐₘₚₗₑ (Sample volume in reaction) = 0.03 mL (30 µL)T (Reaction time) = 5 minCpr (Sample protein concentration, mg/mL)W (Sample mass, g)Vₛₐₘₚₗₑₜₒₜₐₗ (Total extract volume) = 1.0 mL (for tissue/cell calculations)500 (Cell/Bacteria count in millions for example calculation: 5 million)1. For Serum (Plasma):Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per ml of serum.Calculation:HK Activity (nmol/min/ml) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ TSimplified Formula: HK (nmol/min/ml) = 1113 × ΔA2. For Tissues, Bacteria, or Cells:a. Based on Sample Protein Concentration:* Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per mg of protein.* Calculation:HK Activity (nmol/min/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ TSimplified Formula: HK (nmol/min/mg prot) = 1113 × ΔA ÷ Cprb. Based on Sample Fresh Weight:* Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per gram of fresh tissue.* Calculation:HK Activity (nmol/min/g fresh weight) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (W × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: HK (nmol/min/g fresh weight) = 1113 × ΔA ÷ Wc. Based on Bacterial or Cell Density:* Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per 10⁴ cells.* Calculation (example for 5 million cells in 1 ml extract):HK Activity (nmol/min/10⁴ cell) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (500 × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: HK (nmol/min/10⁴ cell) = 2.226 × ΔAPrecautionsPerform a pilot assay with 2-3 samples expected to have significant activity differences before the formal determination... Read More | Annexin V ( annexin-V ) is a Ca2 + dependent phospholipid binding protein with a molecular weight of 35-36 KD, which can selectively bind to phosphatidylserine ( PS ). Phosphatidylserine ( PS ) is mainly distributed in the inner side of the cell membrane, that is, the side adjacent to the cytoplasm.Annexin V ( annexin-V ) is a Ca2 + dependent phospholipid binding protein with a molecular weight of 35-36 KD, which can selectively bind to phosphatidylserine ( PS ). Phosphatidylserine ( PS ) is mainly distributed in the inner side of the cell membrane, that is, the side adjacent to the cytoplasm. In the early stage of apoptosis, different types of cells will turn phosphatidylserine out to the cell surface and expose to the extracellular environment. At this time, using Annexin V labeled with fluorescent protein PE, that is, Annexin V-PE, combined with phosphatidylserine ( PS ), the eversion of phosphatidylserine, an important feature of apoptosis, can be directly detected by flow cytometry. Normal cells will not be stained by Annexin V-PE, apoptotic or necrotic cells will be stained by Annexin V-PE. Annexin V-PE can be used in combination with partially non-permeable nuclear dye ( 7-AAD / PI ) to distinguish cells at different stages of apoptosis. RedNucleus II provided in this kit is a far-red dye that belongs to an anthraquinone compound and cannot penetrate the intact cell membrane of living cells and early apoptotic cells. It is non-permeable, but can quickly stain the nucleus / dsDNA in dead and permeable cells. RedNucleus II is an ideal substitute for propidium iodide ( PI ) and 7-AAD.Combined with Annexin V-PE, it has better spectral characteristics without compensation regulation : it is not excited by ultraviolet light and does not overlap with PE / PE homologues, so it can be combined with FITC, PE and purple fluorescent dyes for multicolor analysis. When combined with Annexin V-PE, RedNucleus II was excluded from living cells and early apoptotic cells, while late apoptotic cells and dead cells were double-positive for Annexin V-PE and RedNucleus II. Annexin V-PE / RedNucleus II apoptosis detection kit can be detected by flow cytometry or other fluorescence detection equipment. Components: Components A598354(10T) A598354(50T) A598354(100T) A. 1×Annexin V Combining buffer solution 10 mL 50 mL 50 mL×2 B. Annexin V-PE 50 µL 250 µL 500 µL C. RedNucleus II 100 µL 500 µL 1 mLProduct parameters：Annexin v-pe:ex/em=488/578 nmrednucleus ii:ex/em=635/695 NMUsage method:1. Experimental design: Blank tube: Negative control group cells, without Annexin V-PE/RedNucleus II. Used to regulate voltage.Single staining tube: Positive control group cells were treated with Annexin V-PE alone/RedNucleus II alone. Used for adjusting compensation.Detection tube: Add Annexin V-PE/RedNucleus II to the processed cells. After adjusting the voltage compensation using blank tubes and single dye tubes, obtain the required flow data.2. Collect cells(1) For suspended cells:a. After inducing cell apoptosis, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS, and count them.Note: PBS resuspension cannot be omitted. The process of PBS resuspension also serves to wash cells, ensuring the subsequent binding of Annexin V-PE.b. Take 5 × 104-1 × 105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 100 µ L of 1 × Annexin V binding buffer to gently resuspend the cells. c. Add 5 µ L Annexin V-PE and mix gently.d. Add 5 µ L of RedNucleus II staining solution and mix gently.e. Incubate at room temperature (20-25 º C) in the dark for 15 minutes. Aluminum foil can be used to avoid light. During the incubation process, cells can be resuspended 2-3 times to improve staining efficiency.(2) For adherent cells:a. Suck out the cell culture medium into a suitable centrifuge tube, wash the adherent cells with PBS once, and add an appropriate amount of trypsin cell digestion solution (without EDTA) to digest the cells. Incubate at room temperature until gently blowing can remove the trypsin cell digestion solution when the adherent cells are blown down. Overdigestion of pancreatic enzymes should be avoided.Note: For adherent cells, the trypsin digestion step is crucial. If the trypsin digestion time is too short, cells need to be blown hard to detach, which can easily cause damage to the cell membrane and lead to false positives of cell necrosis; If the digestion time is too long, it can also cause cell membrane damage and false positives of cell necrosis, and even affect the binding of phosphatidylserine and Annexin V-PE on the cell membrane, thereby interfering with the detection of cell apoptosis.b. Add the cell culture medium collected in the previous step, gently blow down the cells, transfer them to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count them.Note: Adding the cell culture medium from the previous step is very important. On the one hand, it can collect cells that have already been suspended and undergone apoptosis or necrosis. On the other hand, the serum in the cell culture medium can effectively inhibit or neutralize residual trypsin. The residual trypsin will digest and degrade the subsequently added Annexin V-PE, leading to staining failure.c. Take 5 × 104-1 × 105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 100 µ L of 1 × Annexin V binding buffer to gently resuspend the cells. d. Add 5 µ L Annexin V-PE and mix gently.e. Add 5 µ L of RedNucleus II staining solution and mix gently.f. Incubate at room temperature (20-25 º C) in the dark for 15 minutes. Aluminum foil can be used to avoid light. During the incubation process, cells can be resuspended 2-3 times to improve staining efficiency.3. Result analysis:(1) Flow cytometry detection:a. After incubation, 400 µ L of 1 × Annexin V binding buffer can be directly added to resuspend the cells, and immediately detected on the machine. Annexin V-PE is excited by 488 nm/566 nm laser, and the fluorescence emission spectrum is detected at 578 nm (BL2 (FL2)/YL1 channel), while the RedNucleus II channel emission spectrum is approximately at 695 nm (RL1 (FL4) channel).b. On the scatter plot of the bivariate flow cytometer, live cells are shown in the lower left quadrant, which is (Annexin V-PE -/RedNucleus II -); The lower right quadrant represents early apoptotic cells, which are (Annexin V-PE+/RedNucleus II -); The upper right quadrant represents necrotic and late stage apoptotic cells, which are (Annexin V-PE+/RedNucleus II+); The upper left quadrant displays naked nuclear cells, which are (Annexin V-PE -/RedNucleus II+).(2) Fluorescence microscopy detection:a. Centrifuge at 1000 rpm for 5 minutes, collect cells, and gently resuspend them in 400 µ L of 1 × Annexin V binding buffer. Transfer the cells to a 96 well plate and settle for a moment or perform cell smear, then observe under a fluorescence microscope.b. Annexin V-PE is compatible with PE filters. RedNucleus II can use a far red long pass filter.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. to reduce the process of apoptosis, the incubation process can be operated on ice, but the incubation time should be extended to at least 30 min. 3. as apoptosis is a rapid process, it is recommended that samples be analyzed within 1 h after staining. 4. for adherent cells, digestion is a key step. If there are floating cells when adherent cells induce apoptosis, the floating cells and adherent cells should be collected and stained. Handle adherent cells with care to avoid artificial damage to cells. The trypsin digestion time is too short, and the cells need to be blown hard to fall off, which is easy to cause damage to the cell membrane and excessive intake of rednucleus II; If the digestion time is too long, the cell membrane is also prone to damage, and even affect the binding of phosphatidylserine and annexin v-pe on the cell membrane. When digesting, spread pancreatin on the bottom of the well plate, fully contact the pancreatin with the cells when shaking gently, then pour out most of the pancreatin, use the remaining small amount of pancreatin to digest for a period of time, and terminate when the gap between cells increases and the bottom of the bottle is spotted. Try not to use EDTA in the digestive juice, which will affect the binding of annexin V to PS. 5. after the adherent cells are digested with trypsin, it is recommended to stain after recovering in the optimal culture conditions and medium for about 30 min to avoid false positives. 6. in order to avoid losing cells when washing cells, you can use a large tip over a small tip to aspirate. 7. the optimal concentration of dye is determined by the specific experimental requirements. 8. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 9. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Early apoptosis detection, annexin V Kit... Read More | Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) uses RedNucleus I staining to detect cell cycle. RedNucleus I is a far-infrared nucleic acid dye with cell membrane permeability, which can quickly enter living cells, specifically bind to DNA, and perform cell cycle detection on living cells without RNase digestion. Compared with the traditional PI staining method, the cells do not need to be broken or fixed, and the operation is simpler. RedNucleus I is a fluorescent dye of double-stranded DNA, and the fluorescence intensity after binding to double-stranded DNA is proportional to the content of double-stranded DNA. The intracellular DNA content can be measured by flow cytometry, and then the cell cycle analysis can be carried out according to the distribution of DNA content. After RedNucleus I staining, assuming that the fluorescence intensity of G0 / G1 phase cells is 1, the theoretical value of the fluorescence intensity of G2 / M phase cells containing two copies of genomic DNA is 2, and the fluorescence intensity of S phase cells undergoing DNA replication is between 1-2. In addition, RedNucleus I is compatible with dyes such as Horizon BV / BUV, FITC and R-PE, and can be periodically detected after sample staining.The kit is usually used to detect the cell cycle of cultured adherent or suspended cells. If it is used for cell cycle detection of tissues, the tissues must be digested into a single cell state.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. this product is applicable to the detection of living cells and fixed cell cycle with certain limitations. Whether it is applicable to different types of cells needs to be determined after testing. If fixation is needed, it is recommended to use ice bath pre cooling 75-80% ethanol -20 ℃ to fix cells overnight. 3. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Instruction： Experimental materials ( self-provided )：①cell lines or other cell samples ( self-prepared ) ；②This kit ; ③ trypsin ( self-prepared ) ;④ Cell culture medium containing FBS ( self-prepared ) ; Experimental procedure： 1.Preparation of cell samples : ( 1 ) ( This step is for adherent cells, if suspended cells, can be carried out directly step ( 2 ) ) Digest cells with trypsin, add cell culture medium, gently blow away cells, collected into the centrifuge tube. Note : The number of cells on the machine needs to reach 50,000 and above, so the initial number of cells collected needs to be sufficient. ( 2 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. Carefully remove the supernatant, add about 1 mL of ice bath pre-cooled 1 × staining buffer ( 10 × staining buffer diluted with diH2O at 1 : 10 ), re-suspend the cells. Repeat once. ( 3 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. After the supernatant was discarded, 1 mL of culture medium was added to re-suspend the cells ( for fixed cells, 1 × PBS can also be used to re-suspend ). Gently flick the bottom of the centrifuge tube to properly disperse the cells to avoid cell aggregation. 2.Staining : 4 µL of RedNucleus I staining solution was added to each tube of cell samples, slowly and fully mixed, and incubated at room temperature in dark for 20 min ( or incubated at 37 ° C in dark for 5-10 min ). The optimal incubation time of different cells is different, and the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect. 3.Flow cytometry detection and analysis : Excited at 638 nm by flow cytometry, it is recommended to detect in RL3 or FL4 channels, or use RL1 and RL2 channels. Cell DNA content analysis and light scattering analysis were performed using appropriate analysis software.Scope of application：Cell cycle detection... Read More | DescriptionPhoto KitAlysis Starter Kit enables screening of 24 micro-scale simultaneous photocatalytic reactions with consistent and reproducible photon intensity. User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsComponents:Photo KitAlysis LED ControllerBlue LED DescriptionPhoto KitAlysis Starter Kit enables screening of 24 micro-scale simultaneous photocatalytic reactions with consistent and reproducible photon intensity. User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsComponents:Photo KitAlysis LED ControllerBlue LED Array (470 nm)Photo KitAlysis Reaction BlockTorque screwdriverSmall screwdriver to easily remove torqued screws after reaction is completeFeatures:Designed and tested by synthetic chemists.Controller provides repeatable milliamp selection for photon intensity0-30 mA variable LED output3 different LED options: blue (470 nm, included), green (527 nm, sold separately), and white (sold separately)Non-magnetic LED baseChemically resistant LED coverPTFE coated cablingDesigned to be used withPhoto KitAlysis High-Throughput Reaction Screening Kit(sold separately).Best when used withKitAlysis Benchtop Inertion Box(sold separately)... Read More | The fluorescent dye PKH67 is suitable for conventional cell membrane labeling. It is a green fluorescent dye that can track cells in vitro and in vivo. It labels cells by binding to the lipid components of the membrane structure. PKH67 has low cytotoxicity, low fluorescence background, high fat The fluorescent dye PKH67 is suitable for conventional cell membrane labeling. It is a green fluorescent dye that can track cells in vitro and in vivo. It labels cells by binding to the lipid components of the membrane structure. PKH67 has low cytotoxicity, low fluorescence background, high fat solubility, can easily penetrate cell membranes, and has strong and stable green fluorescence. PKH67-labeled cells can be used for in vitro and in vivo proliferation studies, and have the function of not staining neighboring cells. In the process of cell division and proliferation, the fluorescence intensity of PKH67 will gradually decrease as the cells divide. The labeled fluorescence can be evenly distributed to the two sub-generation cells, so its fluorescence intensity is half that of the parent cell. According to this feature, It can be used to detect cell proliferation, cell cycle estimation and cell division, etc. The fluorescence of PKH67-labeled cells is very uniform, and the fluorescence distribution of sub-generation cells after division is also more uniform. In the process of cell division and proliferation, PKH67-labeled fluorescence can be evenly distributed between the two sub-generation cells, and the fluorescence intensity becomes half of that of the parent cell. According to the difference in fluorescence intensity, the undivided cells can be detected by flow cytometry. One time (1/2 the fluorescence intensity), the second time (1/4 the fluorescence intensity), three times (1/8 the fluorescence intensity), and more divisions of cells. PKH67 can detect splits up to six times or even more. In addition to the detection of cell proliferation, PKH67 can also be used for in vitro tracking of cells. After labeling, the fluorescence expression is stable in the cell, and the positive labeling rate is over 98%. The labeled cells have good morphology, which can effectively observe the cells in vitro. Induce differentiation; or inject labeled cells into the body, it can effectively show the migration and differentiation of transplanted cells in living tissues. PKH67-labeled cells can be used for in vivo observation for as long as several weeks. It is often used for in vivo cell detection experiments and experiments to observe long-term cell activity using fluorescence electron microscope. PKH67 is less toxic and does not affect cell proliferation. This method is simple to operate, does not use radioactive isotopes, and poses no safety hazards. You can get the desired experimental data faster, more accurately and more safely.Due to the longer length of the charcoal tail, internal studies have shown that PKH67 is less transferred between cells than PKH2. In in vivo studies using PKH1 and PKH2, the fluorescence intensity will slowly lose. Since this is a behavioral characteristic of green cell linker dye rather than red cell linker dye, PKH67 will have similar properties. The correlation between the in vitro cell membrane retention of non-dividing cells and the in vivo fluorescence half-life reveals that the in vivo fluorescence half-life of PKH67 is 10-12 days. Other green cell linker dyes with similar half-lives have been used to monitor the transport of lymphocytes and macrophages in the body within one to two months. The results indicate that PKH67 can also be used for medium-term in vivo tracking studies.The dye can stably bind to the lipid region of the cell membrane and emit fluorescence, and is mainly used for cell labeling in vitro, cell proliferation research in vitro, and cell tracing research in vivo and in vitro. The fluorescence half-life of PKH67 in vivo is 10-12 days. Compared with PKH-67, PKH-26 has a longer half-life, and the half-life of PKH26 labeled on rabbit red blood cells is more than 100 days. Especially suitable for in vitro proliferation research and long-term in vivo cell tracking research. After PKH67 labels the cells, flow cytometry is usually used for cell proliferation detection.Kit components0.1ml kits: P266290A-0.1ml P266290B-10ml1ml kits: P266290A-1ml P266290B-60mlDyes with A suffix and diluents with B suffix are used togetherPKH67 labeled cells show green fluorescence, the fluorescence wavelength: λex=490 nm, λem=502 nm.Storage conditions: -20℃ protected from light, valid for 1 yearPrecautions●Staining concentration varies according to the type of cell and the number of cells in each well.● The prepared PKH67 mother liquor is very easy to dissolve. It is recommended to store in aliquots and freeze-dry at ≦-20℃.● PKH67 working solution should be prepared for immediate use, and cannot be prepared in advance, because PKH67 will decompose due to the absorption of water and affect the dyeing effect.● PKH67 is easily decomposed and will deteriorate quickly in the water solution. Please avoid contact with water during use of mother liquor. The working fluid is in contact with the water during the process of labeling the cells within the permitted time range.● PKH67 fluorescent dye is a DMSO solution. It will solidify and stick to the bottom, wall or cap of the tube at a lower temperature such as 4℃ and ice bath. After being taken out of the refrigerator, it will return to room temperature and become After the liquid is in the state, remove the cap from the bottom of the tube. It can be used after it has completely melted in a 37°C water bath.● The number of generations or time that can be traced after different cell types are marked is quite different. Please make a test based on the actual situation or reference documents.Instructions1. Staining solution preparation:(1) Take out the PKH67 reagent from the refrigerator, let it stand for a few minutes to room temperature, or after a 37°C water bath, leave the tube containing PKH67, and be sure to leave the tube for a few minutes before opening the lid to allow the reagent to fully fall into the tube The lid can only be opened after the bottom.(2) According to the number of cell samples to be tested, dilute the probe 10 times with the diluent, and then use a suitable solution (such as non-clear medium, HBSS or PBS) to dilute the PKH67 mother liquor 25 times to prepare a stain Work fluid. The best working solution concentration should be adjusted according to different cells and your own experimental system. Generally, the cells can be diluted 250 times according to the final concentration of the mother liquor in the kit. Some cells may need to increase the concentration appropriately.2. Cell staining(1) Resuspend the prepared cells to be tested in 100µl of staining solution to a cell concentration of about 107/ml. You can also perform in-situ staining, as long as the staining solution is enough to cover the cells.(2) Culture the cells at 2～8℃ for 15～30 minutes. The best culture time is different for different cells.It is recommended to incubate the labeled cells in the staining solution at 37°C for 5 minutes, and then at 4°C for 15 minutes.Low-temperature incubation can reduce the endocytosis of the dye by the cells, help the dye to label the plasma membrane, and reduce the possibility of the dye localizing to cytoplasmic vesicles.(3) After separation, remove the supernatant, collect the cells, wash the cells 1-2 times with PBS or non-clear medium, and finally add PBS or non-clear medium to resuspend the cells.(4) Take 500µl of cell suspension and test with flow cytometer. Ex/Em=490/502nm.(5) Subsequently, the cells can be cultured according to the normal culture method.(6) The labeling effect can be directly observed under a fluorescence microscope, or the cell proliferation can be detected by a flow cytometer after an appropriate period of culture, or used for cell fluorescence traces for other specific experimental purposes... Read More |