| Description | Inquire | 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 | 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 | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Q665720 Component 200T Storage Q665720A Buffer L2 25 mL RT Q665720B Buffer N3 80 mL RT Q665720C Buffer PB 35 mL RT Q665720D Buffer PW (concentrate) 25 mL RT Q665720E Buffer EB 30 mL RT Q665720F RNase A (10 mg/mL) 800 渭L RT Q665720G Spin Columns DM with Collection Tubes 200 EA RTProduct Q665720 Component 200T Storage Q665720A Buffer L2 25 mL RT Q665720B Buffer N3 80 mL RT Q665720C Buffer PB 35 mL RT Q665720D Buffer PW (concentrate) 25 mL RT Q665720E Buffer EB 30 mL RT Q665720F RNase A (10 mg/mL) 800 渭L RT Q665720G Spin Columns DM with Collection Tubes 200 EA RTProduct IntroductionThe biggest feature of this kit: simple and fast, high extraction volume. The whole extraction process does not take more than 10 minutes, without centrifugation to collect bacteria and resuspend the bacterium, directly add the unique super lysate Buffer L2 to the cultured bacterial solution, followed by neutralization, centrifugation and passing through the column, and the extracted plasmid can be as high as 30 µg, and maximize the removal of proteins, genomes and other impurities. The extracted plasmid DNA can be directly used for bacterial transformation, digestion, PCR, in vitro transcription, sequencing and other downstream experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. The kit can be stored in a dry, room temperature (15-30°C) environment for 1 year. For longer storage, the centrifuge columns can be placed at 2-8°C.2. Before the first use, add all of the RNase A solution to Buffer N3, mix well, and store at 2-8°C.3. Anhydrous ethanol should be added to Buffer PW before the first use according to the instructions on the reagent bottle label.4. If there is any precipitation in Buffer L2 before use, please put it in a 37℃ water bath and keep mixing until the solution becomes clear before use.Operation steps1. Take 600 µl of bacterial culture into a 1.5 ml centrifuge tube (supplied).2. Add 100 µl of Buffer L2 to the above centrifuge tube and gently turn the solution up and down 8 times; the solution should change from turbid to a clear purple color, indicating complete lysis. The cleavage time should not exceed 2 minutes.3. Add 350 µl of Buffer N3 to the above centrifuge tube (please check that RNaseA has been added first) and immediately mix well by turning up and down about 8-10 times, at which point the solution should turn completely yellow and a yellow precipitate should form. centrifuge at 13,000 rpm for 2-3 minutes.4. Slowly pour the supernatant obtained in step 3 into the prepared adsorption columns (Spin Columns DM with Collection Tubes) to avoid sedimentation into the columns.5. Centrifuge at 13,000 rpm for 15 seconds, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.6. Add 150 µl Buffer PB to the adsorption column and centrifuge at 13,000 rpm for 15 seconds.7. Add 400 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first) and centrifuge at 13,000 rpm for 1 minute.8. Place the adsorbent column in a new centrifuge tube (self-provided), add 30-100 µl Buffer EB to the middle part of the adsorbent membrane, centrifuge at 13,000 rpm for 1 min, collect the plasmid DNA, and store at -20°C for long term storage.When the amount of extracted bacterial liquid is >600µl, the following procedure can be used:1. This kit can extract up to 3ml of bacterial solution, if the amount of extracted bacterial solution is more than 600µl, it is necessary to centrifuge the bacterial solution exceeding 600µl at 13,000rpm for 30 seconds (to collect the bacterial body), discard the supernatant and then add 600µl of bacterial solution, and then resuspend the bacterial body at the bottom of the tube thoroughly and then proceed to the following operation.2. Add 100µl Buffer L2 to the above centrifuge tube, gently invert the solution up and down 10 times, if the solution is not clarified, need to continue to invert the mixing until the solution becomes a clear purple color, the lysis time should not be more than 2 minutes. (If the solution is still turbid, the amount of bacteria is too large, and the amount of bacteria should be reduced appropriately.)3. Add 350 µl of Buffer N3 to the above centrifuge tube (please check that RNaseA has been added first) and immediately mix well by turning up and down until the purple solution turns completely yellow and a yellow precipitate is formed before proceeding to the next step. centrifuge at 13,000 rpm for 5 minutes.4. Transfer the supernatant to a new centrifuge tube, add 200 µl of isopropanol, mix up and down several times, mix well and transfer to the adsorbent column (Spin Columns DM with Collection Tubes), due to the amount of solution is too large, this time, it is necessary to centrifuge the column in two separate times, centrifugation at 13,000 rpm for 15 seconds, pour off the waste liquid in the collection tube, and put the adsorbent column back to the The adsorbent column should be placed back into the collection tube.5. Add 150 µl Buffer PB to the adsorption column and centrifuge at 13,000 rpm for 15 seconds.6. Add 400 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first) and centrifuge at 13,000 rpm for 1 minute.7. Place the adsorbent column in a new centrifuge tube (self-provided), add 50-200 µl Buffer EB to the middle part of the adsorbent membrane, leave it at room temperature for 2 min, centrifuge at 13,000 rpm for 1 min, collect the plasmid DNA, and store it at -20°C for a long time... Read More |