ab176753 CytoPainter PhalloidiniFluor 488 Reagent Instructions for Use For staining F-actin in adherent or suspension cells. This product is for research use only and is not intended for diagnostic use. Version: 2 Last Updated: 7 April 2014 1 Table of Contents 1. Introduction 3 2. Protocol Summary 5 3. Materials Supplied 5 4. Storage and Stability 6 5. Materials Required, Not Supplied 6 6. Assay Protocol 7 7. Data Analysis 9 8. Troubleshooting 11 2 1. Introduction Actin is a globular, roughly 42-kDa protein found in almost all eukaryotic cells. It is also one of the most highly conserved proteins, differing by no more than 20% in species as diverse as algae and humans. Actin is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, and thin filaments, part of the contractile apparatus in muscle cells. Thus, actin participates in many important cellular processes including muscle contraction, cell motility, cell division and cytokinesis, vesicle and organelle movement, cell signaling, as well as the establishment and maintenance of cell junctions and cell shape. CytoPainter Phalloidin-iFluor 488 Reagent (ab176753) is a green fluorescent phalloidin conjugate (equivalent to Alexa Fluor® 488labeled phalloidin) that selectively binds to actin filaments (also known as F-actin). Used at nanomolar concentrations, phalloidin derivatives are convenient probes for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments. Phalloidin binds to actin filaments much more tightly than to actin monomers, leading to a decrease in the dissociation rate of actin subunits from filament ends, essentially stabilizing actin filaments through the prevention of filament depolymerization. Moreover, phalloidin is found to inhibit the ATP hydrolysis activity of F-actin. Phalloidin functions differently at 3 various concentrations in cells. When introduced into the cytoplasm at low concentrations, phalloidin recruits the less polymerized forms of cytoplasmic actin as well as filamin into stable "islands" of aggregated actin polymers, yet it does not interfere with stress fibers, i.e. thick bundles of microfilaments. Phalloidin is therefore a useful tool for investigating the distribution of F-actin in cells by labeling phalloidin with fluorescent analogs and using them to stain actin filaments for microscopy. Fluorescent derivatives of phalloidin have turned out to be enormously useful in localizing actin filaments in living or fixed cells as well as for visualizing individual actin filaments in vitro. CytoPainter Phalloidin-iFluor 488 Reagent (ab176753) can be detected at Ex/Em = 493/517 nm. Figure 1. Chemical structure of Phalloidin-iFluor 488 Conjugate. 4 2. Protocol Summary Prepare samples in microplate wells Remove liquid from samples in the plate Add phalloidin-ifluor reagent Stain cells at RT for 20-90 min Wash cells Examine the specimen under the microscope 3. Materials Supplied Item Phalloidin-iFluor 488 Conjugate Quantity 1 x 300 tests 5 4. Storage and Stability Upon receipt, store kit at -20°C. Avoid exposure to light. Reagent is stable for at least 6 months if stored properly. Avoid repeated freeze/ thaw cycles. Note: Phalloidin is toxic, although the amount of toxin present in a vial could be lethal only to a mosquito (LD50 of phalloidin = 2 mg/kg), it should be handled with care. 5. Materials Required, Not Supplied PBS with 1% BSA 3-4% formaldehyde in PBS 0.1% Triton X-100 in PBS (optional) Fluorescence microscope equipped with the desired filter set Pipettes and pipette tips Coverslips, petri dishes or well plates to grow cells 6 6. Assay Protocol 1. Reagent Preparation: a) Prepare 1X Phalloidin conjugate working solution by adding 1 µL of 1000X Phalloidin conjugate DMSO solution to 1 mL of PBS with 1% BSA. NOTE: The unused 1000X DMSO stock solution of phalloidin conjugate should be aliquoted and stored at -20 C. protected from light. NOTE: Different cell types might be stained differently. The concentration of phalloidin conjugate working solution should be prepared accordingly. 2. Sample Staining and Analysis: a) Perform formaldehyde fixation. Incubate cells with 3.0–4.0 % formaldehyde in PBS at room temperature for 10–30 minutes. Note: Avoid any methanol containing fixatives since methanol can disrupt actin during the fixation process. The preferred fixative is methanol-free formaldehyde. b) Rinse the fixed cells 2–3 times in PBS. c) Optional: Add 0.1% Triton X-100 in PBS into fixed cells (from Step b) for 3 to 5 minutes to increase permeability. Rinse the cells 2–3 times in PBS. 7 d) Add 100 μL/well (96-well plate) of phalloidin conjugate working solution (from Step 1c) into the fixed cells (from Step 2b or 2c), and stain the cells at room temperature for 20 to 90 minutes. e) Rinse cells gently with PBS 2 to 3 times to remove excess phalloidin conjugate before plating, sealing and imaging under microscope. 8 7. Data Analysis Figure 2. HeLa cells were stained with mouse anti-tubulin followed with a fluorescent red Goat Anti-Mouse IgG, actin filaments were stained with CytoPainter Phalloidin-iFluor 488 Reagent (ab176753), and nuclei were stained with Hoechst 33342. 9 Figure 3. Excitation and emission spectra of CytoPainter PhalloidiniFluor 488 Reagent (ab176753). 10 8. Troubleshooting Problem Reason Solution Too low dye concentration or incubation time insufficient Increase concentration or incubation time Cells observed at incorrect wavelength Ensure you are using appropriate filter settings Cells do not appear healthy Cells require serum to remain healthy Add serum to stain and wash solutions. Try range 2 – 10% serum. Nuclear counterstain is too bright Different microscopes, cameras and filters may make some signals appear very bright Reduce concentration of nuclear counterstain or shorten exposure time. Lysosomes not sufficiently stained. 11 12 13 14 UK, EU and ROW Email: technical@abcam.com | Tel: +44-(0)1223-696000 Austria Email: wissenschaftlicherdienst@abcam.com | Tel: 019-288-259 France Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96 Germany Email: wissenschaftlicherdienst@abcam.com | Tel: 030-896-779-154 Spain Email: soportecientifico@abcam.com | Tel: 911-146-554 Switzerland Email: technical@abcam.com Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530 US and Latin America Email: us.technical@abcam.com | Tel: 888-77-ABCAM (22226) Canada Email: ca.technical@abcam.com | Tel: 877-749-8807 China and Asia Pacific Email: hk.technical@abcam.com | Tel: 108008523689 (中國聯通) Japan Email: technical@abcam.co.jp | Tel: +81-(0)3-6231-0940 www.abcam.com | www.abcam.cn | www.abcam.co.jp 15 Copyright © 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. Copyright © 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.