| Description | Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner. In early-stage apoptosis, the plasma membrane excludes viability dyes such as 4',6-diamidino-2-phenylindole （DAPI）, propidium iodide (PI), 7-AAD. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative). This kit is suitable for the identification and enumeration of dead cells, such as apoptotic or necrotic cells, by flow cytometry.A1456541Components20 T50 T100 TStorageQuantity Per TestA1456541A10× Annexin V Binding Buffer5 mL10 mL20 mL2-8℃200 µL per 0.5-1.0 × 10⁵ cellsA1456541BAnnexin V -PE-Cy7200 µL500 µL1000 µL2-8℃. Store in the dark.10 µL per 0.5-1.0 × 10⁵ cellsA1456541CDAPI Staining Solution (25 µg/mL)20 µL50 µL100 µL2-8℃. Store in the dark.1 µL per 0.5-1.0 × 10⁵ cellsNote: The recommended number of cells to stain per test is 0.5-1.0 × 10⁵ cells.Precautions1. Please try to avoid light when using to slow down the quenching of fluorescence.2. DAPI Solution is toxigenic and mutagenic; handle with care.3. Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments.Instruction for use 1. Dilute 10X Binding Buffer to 1X using distilled water (1 mL 10X Binding Buffer + 9 mL ddH2O).2. Wash cells twice with cold PBS and then resuspend the desired amount of cells in Annexin V Binding Buffer at a concentration of 0.5-1.0x 10⁶ cells /mL. 3. Add 10 µL of Annexin V-PE-Cy7 and 1 µL of DAPI staining solution to 100 µL of cell suspension.4. Add 100 µL of 1x Binding Buffer to each assay. Gently vortex the cells and incubate for 10 min at RT (25°C) in the dark.5. Perform flow cytometry analysis within 1 hr... 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 | DescriptionCholesteryl ester transfer protein (CETP) is present in normal human plasma and transfers neutral lipids from high density lipoproteins (HDL) to very low density lipoprotein (VLDL) and low density lipoprotein (LDL). CETP plays an important role in lipoprotein metabolism and influences theDescriptionCholesteryl ester transfer protein (CETP) is present in normal human plasma and transfers neutral lipids from high density lipoproteins (HDL) to very low density lipoprotein (VLDL) and low density lipoprotein (LDL). CETP plays an important role in lipoprotein metabolism and influences the reverse cholesterol transport pathway.Preparation instructionsSuitable for high-throughput screening (HTS), mechanism of action (MOA) studies, and structure-activity relationship (SAR) work in CETP sources.PrincipleThe CETP RP Activity Assay uses a proprietary substrate that enables the detection of CETP-mediated neutral lipid mass transfer. The method is useful for measuring CETP activity in recombinant protein (RP) or purified CETP samples and has a high D... Read More | Inquire | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export |