ab TMRE Mitochondrial Membrane Potential Assay Kit

Transcription

1 ab TMRE Mitochondrial Membrane Potential Assay Kit Instructions for Use For the measurement of mitochondrial membrane potential by flow cytometry, fluorescence plate reader and fluorescence microscopy. This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 15/07/2014

2 Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY FLOW CYTOMETRY 4 3. ASSAY SUMMARY FLUORESCENCE PLATE READER 5 4. ASSAY SUMMARY FLUORESCENCE MICROSCOPE 6 GENERAL INFORMATION 5. PRECAUTIONS 7 6. STORAGE AND STABILITY 7 7. MATERIALS SUPPLIED 7 8. MATERIALS REQUIRED, NOT SUPPLIED 8 9. LIMITATIONS TECHNICAL HINTS 9 ASSAY PREPARATION 11. REAGENT PREPARATION 10 ASSAY PROCEDURE 12. ASSAY PROCEDURE 11 DATA ANALYSIS 13. TYPICAL DATA 15 RESOURCES 14. FREQUENTLY ASKED QUESTIONS NOTES 18 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Abcam s TMRE Mitochondrial Membrane Potential Assay Kit is designed for interrogating mitochondrial membrane potential in live cells for analysis by flow cytometry, microplate spectrophotometry and fluorescent microscopy. Each assay kit contains sufficient materials for at least 200 measurements. Mitochondrial membrane potential (Δψm) is highly interlinked to many mitochondrial processes. The Δψm controls ATP synthesis, generation of ROS, mitochondrial calcium sequestration, import of proteins into the mitochondrion and mitochondrial membrane dynamics. Conversely, Δψm is controlled by ATP utilization, mitochondrial proton conductance, respiratory chain capacity and mitochondrial calcium. Hence pharmacological changes in Δψm can be associated with a multitude of other mitochondrial pathological parameters which may require further independent evaluation. Depolarization can be found in the presence of ionophores that could induce nonselective cation channels or become selective mobile ionic carriers. Protonophores such as FCCP and CCCP induce reversal of the ATPase, as a compensatory mechanism that tries to maintain Δψm, which will deplete ATP even in the presence of a normal glycolytic pathway. Hyperpolarization could be found in the presence of ATPase inhibition, inadequate supply of ADP, increased supply of NADH, apoptosis due to oxidative stress and potentially proton slippage due to cytochrome c oxidase dephosphorylation. In either scenario, OXPHOS uncoupling ensues. The TMRE mitochondrial membrane potential kit uses TMRE (tetramethylrhodamine, ethyl ester) to label active mitochondria. TMRE is a cell permeant, positively-charged, red-orange dye that readily accumulates in active mitochondria due to their relative negative charge. Depolarized or inactive mitochondria have decreased membrane potential and fail to sequester TMRE. Discover more at 2

4 INTRODUCTION FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) is a ionophore uncoupler of oxidative phosphorylation. Treating cells with FCCP eliminates mitochondrial membrane potential and TMRE staining. TMRE is suitable for the labelling of mitochondria in live cells and is not compatible with fixation. Discover more at 3

5 INTRODUCTION 2. ASSAY SUMMARY FLOW CYTOMETRY Seed cell line(s) and add treatment for desired amount of time Optional: 10 minutes before adding TMRE (next step), add 20 µm FCCP to one sample in media Add TMRE to cells in media to a final concentration of nm. Incubate for 20 minutes at 37 ºC Measure TMRE staining of mitochondria in live cells. Collect data from single cell suspension in media or 0.2% BSA in PBS. (peak excitation=549 nm, peak emission=575 nm) Discover more at 4

6 INTRODUCTION 3. ASSAY SUMMARY FLUORESCENCE PLATE READER Seed cell line(s) and add treatment for desired amount of time Optional: 10 minutes before adding TMRE (next step), add 20 µm FCCP to one sample in media Add TMRE to cells in media to a final concentration of 200 1,000 nm. Incubate for 20 minutes at 37 ºC Measure TMRE staining of mitochondria in live cells. Wash cells once with 0.2% BSA in PBS, then read cells in a microplate. (peak excitation=549 nm, peak emission=575 nm) Discover more at 5

7 INTRODUCTION 4. ASSAY SUMMARY FLUORESCENCE MICROSCOPE Seed cell line(s) and add treatment for desired amount of time Optional: 10 minutes before adding TMRE (next step), add 20 µm FCCP to one sample in media Add TMRE to cells in media to a final concentration of nm. Incubate for 20 minutes at 37 ºC Measure TMRE staining of mitochondria in live cells. Wash cells once in PBS, then image live cells. (peak excitation=549 nm, peak emission=575 nm) Discover more at 6

8 GENERAL INFORMATION 5. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 6. STORAGE AND STABILITY Store kit at 4ºC immediately upon receipt. For longer term storage, keep at -20 C in the dark. Avoid freeze/thaw cycles. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in section MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) 1 mm TMRE in DMSO (~1,000X) 40 µl 4ºC 50 mm FCCP in DMSO (~2,500X) 10 µl 4ºC Discover more at 7

9 GENERAL INFORMATION 8. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: Fluorescence plate reader or Flow cytometer. Required excitation/emission wavelengths: 485 nm/535 nm General tissue culture supplies Sterile PBS (1.4 mm KH2PO4, 8 mm Na2HPO4, 140 mm NaCl, 2.7 mm KCl, ph 7.4) Bovine Serum Albumin (BSA) 9. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures Do not use kit or components if it has exceeded the expiration date on the kit labels Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding Discover more at 8

10 GENERAL INFORMATION 10. TECHNICAL HINTS Clear bottom, dark sided microplates are recommended with this assay. Clear sided microplates have not been tested with this kit TMRE is a live cell stain and is not compatible with fixation. TMRE is light sensitive. Maintain TMRE solutions and TMRE labeled cells in the dark. This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions Discover more at 9

11 ASSAY PREPARATION 11. REAGENT PREPARATION Equilibrate all reagents to room temperature (18-25 C) prior to use. The TMRE provided is sufficient for at least 200 assays (flow cytometry, microplate or microscopy) Working TMRE Solution Prepare a working TMRE solution by adding the appropriate volume of 1 mm TMRE in appropriate cell culture media. As an example, to treat cells a final volume of 10 ml cell culture media with 1 µm TMRE, prepare 1mL of 10 µm TMRE (10µL 1 mm TMRE + 1 ml media) and add this to 9 ml of cell culture media containing cells. The exact concentration of TMRE required will depend on the cell line being used. Exact concentrations should be determined on an individual basis by the end user. Typical working concentrations for the different experiment types using Jurkat cell line as follows: Typical working concentrations Experiment Type nm Microplate Flow cytometry Microscopy μm FCCP (Optional Compound) Dilute 50 mm FCCP in appropriate cell culture media to reach a final concentration of 20 µm. Discover more at 10

12 ASSAY PROCEDURE 12. ASSAY PROCEDURE Equilibrate all materials and prepared reagents to 37 C prior to use. It is recommended to assay all standards, controls and samples in duplicate Treat cells of interest with compounds, culture conditions or other manipulation of interest Treatment times can vary depending on the experiment at hand. For example, chemical uncouplers essentially act instantaneously (e.g. FCCP can depolarize mitochondria within minutes). In contrast, treatments that may have a less direct effect on the mitochondrial electron transport chain or that require changes in protein synthesis or activation may take longer to manifest a change in mitochondrial membrane potential Control compound FCCP. Add 20 µm FCCP to cells in media 10 minutes prior to staining with TMRE (step 2). FCCP is an uncoupler that will eliminate the mitochondrial membrane potential and prevent staining by TMRE Add TMRE to cells in culture and incubate for minutes. TMRE should be added to cells in media. An efficient means to do this is to prepare a 10-20X working solution of TMRE in the appropriate media and overlay this to the experimental cultures such that the final concentration is 1X. Return cells to incubator and culture for an additional minutes Data collection and analysis Discover more at 11

13 ASSAY PROCEDURE Microplate assay Removing the culture media is required to eliminate background fluorescence from the media: Suspension cells: Gently pellet the cells by centrifugation and remove the culture media. Resuspend in a like volume of 0.2% BSA in PBS and pellet again. Resuspend in 0.2% BSA in PBS and transfer to microplate Adherent cells: Cells should be seeded in microplates and allowed to adhere prior to the TMRE staining. After TMRE staining, gently aspirate the media and replace with 0.2% BSA in PBS. Repeat Read the plate on a fluorescence plate reader with settings suitable for TMRE (Ex: 549nm, Em: 575nm) Guidelines for cell numbers: For suspension cells, 100, ,000 cells per well in µL should provide sufficient signal. For adherent cells, culture such that the cells are not overly confluent at the time of data collection. The user will need to determine optimal cell densities for the given cell lines Guidelines for TMRE concentration: Dependent on the cell line at hand. Recommending starting concentrations to test are 200 1,000 nm TMRE Flow cytometry Removing media as described above for the microplate reader is not required; however, we do find an enhancement (in some cases up to 40%) of TMRE fluorescence relative to background if the media has been exchanged for 0.2% BSA in PBS. Discover more at 12

14 ASSAY PROCEDURE The user should determine if washing away the media benefits their analysis Before assessing cells by flow cytometry, be certain that the samples are non-aggregated and in a single cell solution. For adherent cells this will require trypsinization or other means to remove cells from their culture vessel Guidelines for cell numbers: Ideally 10,000 cells should be analyzed and cells should not be overly dense during the experiment (<1x106 cells/ml for suspension cells and not overly-confluent for adherent cells) Guidelines for TMRE concentration: Dependent on the cell line at hand. Starting concentrations to test are nm TMRE TMRE is excited by the 488nM laser and should be detected in the appropriate filter channel (peak emission is 575nm). This is commonly FL Microscopy Cells should be plated in a manner consistent with the available microscope setup for live cell imaging. Cells should be imaged as quickly as possible after being removed from the culture conditions as mitochondrial morphology and function is dependent on temperature and cell health. It is recommended to remove media by a PBS rinse before imaging cells to avoid background caused by the media Guidelines for TMRE concentration: nm, as determined by the user. It is recommended to use the least amount of TMRE that gives a reasonably detectable signal Use appropriate filter set for TMRE. Discover more at 13

15 ASSAY PROCEDURE Appropriate controls FCCP serves as a control for depolarized mitochondria (low mitochondrial membrane potential) At minimum, also include control cells that are not stained with TMRE. Ideally both treated and untreated cells should be included as non-tmre stained controls. Discover more at 14

16 DATA ANALYSIS 13. TYPICAL DATA Data provided for demonstration purposes only. Figure 1. Analysis of TMRE staining using a fluorescent plate reader and a microplate. Chart showing mean fluorescent intensity +/- standard deviation from quadruplicate measurements of 400 nm TMRE stained Jurkat cells in a 96-well microplate +/- treatment with FCCP. Discover more at 15

17 DATA ANALYSIS Figure 2. Analysis of TMRE staining by flow cytometry. Flow cytometry histogram of Jurkat cells stained with 100 nm TMRE with (blue) or without (red) treatment with 100 µm FCCP. A. B. Figure 3. Fluroescence microscopy of TMRE labeled mitochondria in live cells. A. HeLa cells (adherent) were cultured on coverslips and stained with 200 nm TMRE for 20 minutes in media, washed briefly with PBS and immediately imaged. B. Jurkat cells (suspension) were stained and washed as above and then transferred to a slide and immobilized under a coverslip for imaging. Discover more at 16

18 RESOURCES 14. FREQUENTLY ASKED QUESTIONS Q. Can I use TMRE to stain isolated mitochondria? A. TMRE staining requires fully intact mitochondria that retain membrane potential. It is not recommended for staining isolated organelles. Q. Can I use TMRE to stain tissue? A. TMRE is best suited for imaging cells grown in a monolayer (e.g. tissue culture cells). Advanced equipment and preparation would be required to image mitochondria in a tissue preparation. Discover more at 17

19 RESOURCES 15. NOTES Discover more at 18

20 UK, EU and ROW Tel: +44-(0) Austria Tel: France Tel: Germany Tel: Spain Tel: Switzerland Tel (Deutsch): Tel (Français): US and Latin America Tel: ABCAM (22226) Canada Tel: China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. RESOURCES 19