(JC-1) 551302 - BD Biosciences
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1. i Wash each sample with 2 0 ml of 1X Assay Buffer RT Wash each sample with 1 0 ml of 1X Assay Buffer RT Y Resuspend each sample in 0 5 ml of 1X Assay Buffer t Analyze by flow cytometry Diagram 1 Overview of Reagent Preparation and JC 1 Staining Protocol This diagram summarizes the reconstitution of the BD MitoScreen Kit components and sample preparation Please refer to the manual for details Usage The BD MitoScreen Kit is compatible with a wide variety of cell model systems and treatment protocols In flow cytometric applications utilizing intact cells JC 1 has typically been used to study relationships between apoptosis and Ay Most of the published literature utilizing the flow cytometry application has been focused on using JC 1 to study the effects of apoptosis on Aw For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com 10 There are numerous ways to induce apoptosis and each researcher may need to optimize protocols for their own experimental system Researchers are strongly encouraged to refer to the extensive literature for information regarding different protocols that have been used to induce apoptosis The optimal protocol for inducing apoptosis may be cell line dependent It is important to note apoptosis may be present in the absence of observed changes in JC 1 fluorescence This is because de2. as monomers resulting in a decrease of red fluorescence The majority of cells in healthy cultures will have a polarized Ay and hence show JC 1 fluorescence in both the FL 1 and FL 2 channels Figure 1 A C It is likely that there will be a small population of cells that has significantly reduced FL 2 fluorescence JC 1 that fluoresces in the FL 1 channel and lacks fluorescence in the FL 2 channel is indicative of depolarized Ay Depolarized Ay indicates altered mitochondrial function which may be due to apoptosis or other cellular processes The percentage of cells showing reduced fluorescence in the FL 2 channel may vary depending on the cell type and culture system However a large percentage of cells with reduced fluorescence in the FL 2 channel in normal or control cultures may indicate that the health of the culture has been compromised For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com Jurkat T Cells Mouse Thymocytes Control Control Staurosporine Camptothecin JC 1 FL 2 JC 1 FL 1 Figure 1 JC 1 Staining in Control and Apoptotic Cells Cells 1 x 10 cells ml were left untreated vehicle only A C or treated with staurosporine 1pm 4 h camptothecin 4 um 4 h or Fas mAb clone Jo2 Cat No 554254 2 pg ml and Protein G 2 pg ml 1 5 h to induce apoptosis D F Cells were stained with JC 1 according to the protocol and analyzed on a BD FACSCalibur as desc
3. bdbiosciences com In some cases the boundaries of the major population may be harder to define making the division between FL 1 bright FL 2 bright and FL 1 bright FL 2 dull more arbitrary as is shown in C In these cases it is particularly critical to define the approximate percentage of apoptotic cells through alternative assays 3 Since apoptotic cells typically lose JC 1 fluorescence in the FL 2 channel the R2 apoptotic cell gate is drawn to encompass the area directly below the R1 gate as shown in Figure 1 A C The accuracy of these gates is best confirmed using the positive induced sample with demonstrable apoptotic cells Figure 1 D F 4 Once the appropriate R1 and R2 gates have been determined using the control and treated samples they should be maintained throughout all samples of equivalent cell types for that experiment on a given day 5 As the user becomes familiar with their experimental system they may wish to set up gating templates that can be individually adjusted for each experiment It should always be kept in mind that the actual Ay JC 1 staining profile seen in a given population control or treated may depend on a variety of factors including the model system the cell type the overall health of the cell culture and treatment type or time For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com References 1 Finkel E 2001 The mitochondrion Is it c
4. be determined by counting cell populations on a hemocytometer Proceed with treatment of choice eg apoptosis induction or leave untreated At the end of the treatment period transfer 1 ml of each cell suspension into a sterile 15 ml polystyrene centrifuge tube Centrifuge cells at 400 x g for 5 minutes at RT Carefully remove and discard the supernatant Add 0 5 ml of freshly prepared JC 1 Working Solution to each pellet Gently resuspend cells in the JC 1 Working Solution Vortex or use a pipette to disrupt any cell to cell clumping Incubate the cells in JC 1 Working Solution for 10 15 min at 37 C in a CO incubator Wash cells twice following incubation see below wash steps are performed at RT 1st wash Add 2 ml of 1x Assay Buffer to each tube and gently resuspend cells Vortex or use a pipette to disrupt any cell to cell clumping Centrifuge cells at 400 x g for 5 min Carefully remove and discard supernatant 2nd wash Add 1 ml of 1x Assay Buffer to each tube and gently resuspend cells Vortex or use a pipette to disrupt any cell to cell clumping Centrifuge the cells at 400 x g for 5 min Gently resuspend each cell pellet in 0 5 ml of 1x Assay Buffer Vortex or use a pipette to disrupt any cell to cell clumping Analyze cells by flow cytometry see Multi Parameter Flow Cytometry Analysis of JC 1 For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com Flow C
5. 0 samples by flow cytometry using the protocol provided Store the unopened vials of JC 1 and 10x Assay Buffer at 2 C 8 C JC 1 4 Amber Vials Lyophilized There is enough JC 1 in each vial for 25 flow cytometry tests JC 1 is supplied in amber vials because it is light sensitive JC 1 is reconstituted prior to use first into a Stock Solution and then into a Working Solution 10x Assay Buffer 60 ml The 10x Assay Buffer is supplied in excess of what is generally needed for 100 flow cytometry tests The 10x Assay Buffer is diluted to 1x prior to use Please refer to the section Kit Reagent Preparation for details on reconstitution of JC 1 and dilution of 10x Assay Buffer and to Diagram 1 for an overview of reagent preparation and subsequent JC 1 staining For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com 10 ml 90m 125 ul 100 ml T T T T T T T T 60 ml gt 125 ul 10X Assay 1X Assay JC 1 vial JC 1 Stock Solution Buffer Buffer DI H20 DMSO lyophilized reconstituted 12 375 ml JC 1 Stock Solution reconstituted 1X Assay Buffer 12 5 ml JC 1 Working Solution for 12 5 ml 25 flow cytometry samples 0 5 ml sample JC 1 Working Solution Cell sample lt 1x106 cells Add 0 5 ml JC 1 Working Solution sample Incubate for 10 15 min 37 C in CO incubator
6. BD MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit Instruction Manual ty BD Catalog No 551302 RJ BD flow cytometers are Class 1 laser products For Research Use Only Not for use in diagnostic or therapeutic procedures 2014 Becton Dickinson and Company All rights reserved No part of this publication may be reproduced transmitted transcribed stored in retrieval systems or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without prior written permission from BD Biosciences Purchase does not include or carry any right to resell or transfer this product either as a stand alone product or as a component of another product Any use of this product other than the permitted use without the express written authorization of Becton Dickinson and Company is strictly prohibited BD BD Logo and all other trademarks are property of Becton Dickinson and Company 2014 BD Table of Contents Tntroduction ccccccccccccccee eee e cece eceeeeeeeeeeeeeneeecneeecseeeeeeteeeteeeneesseees 4 Researching Mitochondrial Roles in Apoptosis and Other Cellular ProGess s eis suste anne kalas OG EERE 4 The BD MitoScreen Kit Application of JC 1 for Flow D o a E E E E E T T 5 General Information Kit Contents Usage and Storage 0 0 008 8 JC 1 4 Amber Vials Lyophilized rornrrrrnrvrnrrnr
7. Coassarizza 2000 Mitochondrial heterogeneity during staurosporine induced apoptosis in HL60 cells Analysis at the single cell and single organelle level Cytometry 40 189 197 Model system HL60 cells treated with staurosporine For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com Notes For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com 19 United States 877 232 8995 Canada 866 979 9408 Europe 32 2 400 98 95 Japan 0120 8555 90 Asia Pacific 65 6861 0633 Latin America Caribbean 55 11 5185 9995 amp BD Becton Dickinson and Company BD Biosciences 2350 Qume Dr San Jose CA 95131 USA US Ordering 855 236 2772 Technical Service 877 232 8995 Fax 800 325 9637 bdbiosciences com answers bd com Rev 3
8. althy non apoptotic FL 1bright FL 2bright R1 and apoptotic FL 1bright FL 2dull R2 cells Each user will have to define the appropriate gates for their experimental model system of apoptosis The following are some guidelines that will help set appropriate gates 1 Given the variability it is critical that whenever possible positive induced apoptotic and control untreated samples be included in every experiment for every cell type In preliminary experiments it is strongly recommended that the presence in the induced sample or absence in the control sample of apoptotic cells be supported by an alternative apoptosis assay such as Annexin V or Active Caspase 3 These products are available in a number of formats please visit www bdbiosciences com for more information on our full line of apoptosis products 2 The R1 non apoptotic cell gate should be set to encompass the major population s in the control sample Figure 1 A C show the gates set for three different experiments The shape and placement of the gate will vary from cell type to cell type Generally there is clearly a single major distinct population of FL 1 bright FL 2 bright cells as in Figure 1 A and B However because the shape and size of the R1 gate can vary on different days direct comparisons between similar cell types using this approach should be carried out on the same day For Research Use Only Not for use in diagnostic or therapeutic procedures www
9. brane polarization or apoptosis For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com The BD MitoScreen Kit contains JC 1 which has been optimized for use in flow cytometry and 10x Assay Buffer JC 1 is typically excited using the 488 nm line of an argon ion laser JC 1 monomers emit maximally at 527 nm and aggregates at 590 nm In the existing literature a number of ways have been used to set up flow cytometers for measurement of the FL 1 and FL 2 channels Factors which can be varied include the PMT settings for each channel and the amount of compensation Data may appear differently depending on how the machine is set up In this manual we provide a standardized approach to allow for reproducible instrument set up Representative data obtained using the approach described herein is shown in Figure 1 A partial list of publications containing figures with additional JC 1 flow cytometry data is provided in the section JC 1 Bibliography Given potential differences in instrument settings data obtained using the standardized set up may appear to be different from previously published data General Information Kit Contents Usage and Storage The BD MitoScreen Kit is designed for use in flow cytometry It consists of two reagents JC 1 and 10x Assay Buffer JC 1 excites at 488 490 nm The monomeric form emits at 527 nm J aggregates emit at 590 nm There are enough reagents to test 10
10. del system human peripheral blood mononuclear cells treated with 2 chloro adenosine and 2 chloro 2 deoxy adenosine 5 Facompre M N Wattez J Kluza A Lansiaux and C Bailly 2000 Relationship between cell cycle changes and variations of the mitochondrial membrane potential induced by etoposide Mol Cell Biol Res Comm 4 37 42 Model system HL 60 cells treated with etoposide 6 Gravance C G D L Garner J Baumber and B A Ball 2000 Assessment of equine sperm mitochondrial function using JC 1 Theriogenology 53 1691 1703 Model system equine spermatozoa For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com 17 18 7 Mathur A Y Hong B K Kemp A A Barrientos and J D Erusalimsky 2000 Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes J Cardiovascular Res 46 126 138 Model system rat cardiomyocytes Mantymaa P T Sitonen T Guttorm M Saily V Kinnula E R Savolainen and P Koistinen 2000 Introduction of mitochondrial manganes superoxide dismutase confers resistance to apoptosis in acute myeloblastic leukemia cells exposed to etoposide British J Haematol 108 574 581 Model system acute myeloblastic leukemia cell line OCI AML 2 subclones treated with etoposide Salvioli S k J Dobrucki L Moretti L Troiano M G Fernandez M Pinti J Pedrazzi C Franceshi and A
11. e of differentiation development normalcy or pathology Two views of the mode of action are emerging For example there is an abundance of data suggesting that mitochondria play a critical role in apoptosis by releasing cytochrome c and other proteins that are essential for the activation of pro caspase 9 and the execution of apoptosis In this scenario mitochondrial activated caspase 9 activates caspase 3 Caspase 3 is often referred to as the primary executioner of apoptosis because it cleaves multiple downstream proteins leading to a loss of cellular structure and function and ultimately cell death Hence one hypothesis supports the view that mitochondria are the primary triggers of cell death rather than the caspases Other data suggest that mitochondria act more as facilitators rather than essential players of apoptosis For example some signals may route to caspase activation without first involving the mitochondria and thereby the activated caspases may target the mitochondria along with other cellular components In this model the caspases would be the primary triggers of cell death and mitochondria along with mitochondrial linked caspase 9 would contribute to cellular demise rather than being essential for it Given the complexity of apoptosis it is likely that there are a number of mechanisms available to the cell for carrying out the process of apoptosis reviewed in 2 Assays designed to evaluate the functional status of mitoch
12. entral to apoptosis Science 292 624 626 Leist M and M Jaattela 2001 Four deaths and a funeral from caspases to alternative mechanisms Nature Rev Mol Cell Biol 2 589 598 Darzynkiewicz Z E Bedner and P Smolewski 2001 Flow cytometry in analysis of cell cycle and apoptosis Seminars Hematol 38 179 193 LeMasters J J A L Nieminen T Qian L C Trost S P Elmore Y Nishimure R A Crowe W E Cascio D A Brenner and B Herman 1998 The mitochondrial permeability transition in cell death common mechanism in necrosis apoptosis and autophagy Biochim Biophys Acta 1366 177 196 Facompre M N Wattez J Kluza A Lansiaux and C Bailly 2000 Relationship between cell cycle changes and variations of the mitochondrial membrane potential induced by etoposide Mol Cell Biol Res Comm 4 37 42 Reers M S T Smiley C Mottola Hartshorn A Chen M Lin and L B Chen 1995 Mitochondrial membrane potential monitored by JC 1 dye Methods Enzymol 260 406 417 Cossarizza A M Baccarani Contri G Kalashnikova and C Franceschi 1993 A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J aggregate forming lipophilic cationic 5 5 6 6 tetrachloro 1 1 3 3 tetraethylbenzimidazolcarbocyanine iodide JC 1 Biochem Biophs Res Comm 197 40 45 For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com Bibliograp
13. hy The following is a partial list of publications containing figures with cells stained with JC 1 and analyzed by flow cytometric analysis 1 Cossarizza A M Baccarani Contri G Kalashnikova and C Franceschi 1993 A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J aggregate forming lipophilic cationic 5 5 6 6 tetrachloro 1 1 3 3 tetraethylbenzimidazolcarbocyanine iodide JC 1 Biochem Biophs Res Comm 197 40 45 Model system U937 and K562 cells treated with valinomycin 2 Petit P X H Lecoeur E Zorn C Dauguet B Mignotte and M L Gougeon 1995 Alterations in mitochondrial structure and function are early events of dexamethasone induced thymocyte apoptosis J Cell Biol 130 157 167 Model system mouse thymocytes treated with dexamethasone 3 Salvioli S Ardizzoni A Franceschi and A Cossarizza 1997 JC 1 but not DiOC6 3 or rhodamine 123 is a reliable fluorescent probe to assess Ay changes in intact cells implications for studies on mitochondrial functionality during apoptosis FEBS 411 72 82 Model system U937 cells treated with ouabain KCl valinomycin or FCCP 4 Barbieri D M P Abbracchio S Salvioli D Monti A Cossarizza S Ceruti R Brambilla F Cattabeni K A Jacobson and C Franceschi 1998 Apoptosis by 2 chloro 2 deoxyadenosine and 2 chloro adenosine in human peripheral blood mononuclear cells Neurochemi Int 32 493 504 Mo
14. n the FL 2 channel R2 and are FL 1 bright FL 2 dull indicative of depolarized Ay These observations are consistent with the notion that mitochondria in control cells are primarily healthy and functioning normally polarized Aw The small percentage of the control population of cells with depolarized Ay may reflect a basal level of apoptosis or presence of other cellular processes that are associated with depolarized Av For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com 13 The fluorescence pattern of JC staining differs dramatically even on healthy control cells Figure 1 A C For example a comparison of the major populations R1 in Figure 1A human Jurkat cells and C primary mouse thymocytes shows that the control Jurkat cells show lower fluorescence intensity in both the FL 1 and FL 2 channels The pattern can even be different using the same cell type on different days compare A and B There is a significant increase in the number of cells with lowered red fluorescence FL 2 R2 in the apoptotic compared to the corresponding control populations compare Figure 1 D F to A C respectively These results are consistent with the notion that apoptosis is often associated with a transition from polarized to depolarized Ay Guidelines for Setting Gates As the pattern of JC 1 staining can vary it is not possible to provide users with universal guidelines for how to set gates for he
15. nrornrrnrnrrnrrrnrnnnnrnnnr 8 10 Assay Butter 60 lun usvssuibndtrtjamniv based samma etterat 8 VUSAPE A T A E derma E ie dnndaundasinnde 9 Kit Reagent Preparation iuie Aa INNAM ES Ernie a 10 Dilution of 10x Assay Buffer rrasrrsrrrrvrnrnvrrrrvnrrrrnrrnnrnrerrrnnrnnnnnnn 10 Preparation Or JC V street Gee tann hse teste tee een 10 Methods for Staining Cells with JC 1 and Analyzing by Flow Cytometny sv 44 s arrene dann 12 Staining Gells with JO Tesseire iane 12 Flow Cytometer Setup ccccccecceeceeteeeeeceeneecteeeeeenteenteneensenneenteees 13 Instrument Setup with BD CaliBRITE Beads 0 0 ceeeeeeees 13 Multi parameter Flow Cytometry Analysis of JC 1 orrrnrrrrnrvnnrn 13 Guidelines for Setting Gates 0 0 ccccccecceeeeceeteeeeeeeenteetetneenteneens 14 Referentes sara eee ened canes Hee eaters 16 Bibliography ren E E 17 Introduction Researching Mitochondrial Roles in Apoptosis and Other Cellular Processes There is a burgeoning interest in the scientific community to fully define the roles of mitochondria in cellular processes particularly apoptosis reviewed in 1 Apoptosis is a complex process that can be induced by many different factors which in turn act through various cell death signaling pathways The role of the mitochondria could potentially vary and may be dependent on a variety of factors including mode of apoptosis induction cell type or cell status with respect to the cell cycle stat
16. ondria are emerging as useful tools for helping to elucidate mitochondrial roles in apoptosis the cell cycle and other cellular processes Particular focus has recently been given to assays designed to study the mitochondrial membrane potential Ay during apoptosis reviewed in 3 Energy released during the oxidation reactions in the mitochondrial respiratory chain is stored as a negative electrochemical gradient across the mitochondrial membrane and the Ay is referred to as being polarized Collapse of the Ay results in a depolarized Ay and is often but not always observed to occur early during apoptosis For example collapse of the Ay during apoptosis has been reported in a number of studies leading to a generalization that depolarization of the mitochondria is one of the first events occurring during apoptosis and may even be a prerequisite for For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com cytochrome c release However this generalization is now a matter of debate and there is data indicating that collapse of the Ay does not always occur during apoptosis Thus depolarization of the Ay may be a cause of or be associated with apoptosis in some but not all systems This is consistent with the concept that there are different mechanisms available for cells to carry out the process of apoptosis In addition to apoptosis changes in the Ay have also been described during necrosis depola
17. polarization of the Ay does not always occur during apoptosis or may not be present at the time point examined Researchers are encouraged to complement their JC 1 studies with other apoptosis assays to gain additional information about the status of their model system Kit Reagent Preparation The 10x Assay Buffer is diluted to a 1x solution prior to use and JC 1 is reconstituted into a Stock Solution and then diluted to a Working Solution prior to use Diagram 1 Dilution of 10x Assay Buffer The Assay Buffer is formulated for use as reaction buffer and for washing the cells It is supplied as a 10x concentrate which must be diluted to 1x with DI H30 prior to use 1 To completely dissolve any salt crystals that may have come out of solution gently warm the 10x Assay Buffer in a 37 C water bath 2 Dilute the 10x Assay Buffer 1 10 in DI H20 For example add 10 ml 10x Assay Buffer to 90 ml DI H30 3 Stir the solution for 5 min 4 Warm the 1x Assay Buffer to 37 C prior to use Note We recommend diluting only the amount of 10x Assay Buffer that will be used in a given day However unused diluted 1x assay buffer may be stored for up to 7 days at 2 C 8 C Preparation of JC 1 JC 1 is supplied lyophilized It is first prepared as a Stock Solution and then used as a Working Solution The JC 1 Stock Solution may be aliquoted and stored at 20 C The Working Solution prepared from the Stock Solution must be used immedia
18. polarized and JC 1 is rapidly taken up by such mitochondria This uptake increases the concentration gradient of JC 1 leading to the formation of JC 1 aggregates known as J aggregates within the mitochondria JC 1 aggregates show a red spectral shift resulting in higher levels of red fluorescence emission which is measured in the Red FL 2 channel on most flow cytometers For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com Although JC 1 fluorescence is seen in both the FL 1 and FL 2 channels in healthy cells the pattern of JC 1 staining may vary between cell type or cell line For example the mitochondria of cardiac muscle cells are more polarized than those in bladder epithelial cells and have brighter red fluorescence In addition to mitochondrial heterogeneity between cell lines intercellular heterogeneity within a cell population may exist Therefore the level of brightness in the FL 2 channel can vary both between and within cell types and lines JC 1 does not accumulate in mitochondria with depolarized Ay and remains in the cytoplasm as monomers These monomers do not have the red spectral shift and therefore have lowered fluorescence in the FL 2 channel The formation of JC 1 aggregates is reversible Thus in mitochondria undergoing a transition from polarized to depolarized Ay due to apoptosis or other physiological events JC 1 leaks out of the mitochondria into the cytoplasm
19. refore 12 5 ml JC 1 Working Solution would be enough for 25 flow cytometry samples 3 Vortex the JC 1 Working Solution thoroughly 4 After vortexing particulate matter consisting of JC 1 aggregates may be present This should not interfere with flow cytometric analysis However if desired the solution can be clarified by centrifugation at 13 000 x g in a microfuge for 3 min or 15 min in a centrifuge at 1 000 x g at RT Transfer the clarified supernatant to a clean tube and discard particulates 5 The JC 1 Working solution must be used immediately after preparation Proceed to the section Methods for Staining Cells with JC 1 and Analyzing by Flow Cytometry for JC 1 staining and analysis protocols Discard leftover JC 1 Working Solution do not store For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com 11 Methods for Staining Cells with JC 1 and Analyzing by Flow Cytometry An overview of staining cells with JC 1 for use in flow cytometry is illustrated in Diagram 1 Details for staining and flow cytometric analysis follow Staining Cells with JC 1 1 Q 10 11 12 13 Culture cells to an optimal density Typically cell density in the cell culture flasks should not exceed 1 x 106 cells per ml Cells cultivated in excess of this concentration may begin to naturally enter apoptosis Optimal cell concentration will vary depending on the cell line used Concentration can
20. ribed in the section Methods for Staining Cells with JC 1 and Analyzing by Flow Cytometry A C JC 1 fluorescence is seen in both the FL 2 and FL 1 channels R1 in the control untreated cell populations A small percentage of the population shows decreased fluorescence in the FL 2 channel R2 D F There is a significant increase in the number of cells with lowered red fluorescence FL 2 R2 indicative of a change in the Ay in the populations induced to undergo apoptosis JC 1 that fluoresces in both the FL 2 and FL 1 channels is considered to correspond to mitochondria with a polarized Ay JC 1 that fluoresces in the FL 1 channel and lacks fluorescence in the FL 2 channel is considered to correspond to mitochondria with a depolarized Ay Thus the data indicates that apoptosis induction was associated with depolarization of the Ay The addition of Protein G enhances the ability of Jo2 to induce apoptosis presumably by cross linking Fas receptors Apoptosis is frequently associated with depolarization of the Ay resulting in increased numbers of cells with reduced JC 1 fluorescence in the FL 2 channel Figure 1 D F That is the apoptotic population frequently presents a lower red fluorescence signal intensity FL 2 axis than the negative control population In some apoptotic systems changes in the level of green fluorescence measured in FL 1 has also been observed It is not clear how these changes relate to changes in the level of mem
21. rization and cell cycle arrest hyperpolarization gt Knowledge of the Ay and how it changes during apoptosis necrosis and the cell cycle may help to clarify the role of the mitochondria in these and other cellular processes The BD MitoScreen Kit Application of JC 1 for Flow Cytometry Flow cytometry has emerged as the technique of choice for analysis of the Ay in whole cells Membrane permeable lipophilic cationic fluorochromes are used as probes of Ay they penetrate cells and their fluorescence is a reflection of Ay JC 1 5 5 6 6 tetrachloro 1 1 3 3 tetraethylbenzimidazolcarbocyanine iodide is a lipophilic fluorochrome that is used to evaluate the status of the Ay reviewed in 6 JC 1 stands for 1st J aggregate forming cationic dye found to be sensitive to the Av The fluorescence emission spectrum of JC 1 is dependent on its concentration which in turn is determined by the status of the Ay JC 1 can exist in two different states aggregates or monomers each with a different emission spectra JC 1 forms monomers at low dye concentrations and aggregates at higher concentrations Both JC 1 aggregates and monomers exhibit fluorescence in the green end of the spectrum which is measured in the Green FL 1 channel on flow cytometers When live cells are incubated with JC 1 JC 1 penetrates the plasma membrane of cells as monomers Uptake of JC 1 into mitochondria is driven by the Ay The Ay of normal healthy mitochondria is
22. tely after preparation and cannot be stored For Research Use Only Not for use in diagnostic or therapeutic procedures www bdbiosciences com Preparation of JC 1 Stock Solution one vial 1 Reconstitute the lyophilized JC 1 reagent at room temperature with 125 pl DMSO per vial to yield a JC 1 Stock Solution Re cap the vial and invert several times to fully dissolve the reagent 2 The Stock Solution must be used immediately by diluting into a Working Solution see Preparation of JC 1 Working Solution from Stock Solution or aliquoted into amber vials to protect against light and stored at 20 C for up to six months 3 Avoid repeated freeze thaws of individual vials of JC 1 Stock Solution as this may compromise its integrity Hence we recommend that when making aliquots of JC 1 Stock Solution for freezing that each aliquot contains only the amount that will likely be needed at the time of thawing Note The kit contains 4 vials of lyophilized JC 1 reagent and we recommend reconstituting only the amount of vials needed for a given experiment Preparation of JC 1 Working Solution from Stock Solution 1 Warm the 1x Assay Buffer to 37 C 2 Prepare a 1x JC 1 Working Solution by diluting the JC 1 Stock Solution 1 100 with prewarmed 1x Assay Buffer For example add 125 pl JC 1 stock to 12 375 ml of prewarmed assay buffer 0 5 ml JC 1 Working Solution is required for each sample cell pellet containing lt 1 x 10 cells The
23. ytometer Setup The Cytometer setup information in this section is designed for the BD FACScan BD FACSort and BD FACSCalibur flow cytometers The BD FACSComp software is used for setting up the flow cytometers Compensation for FL 1 FL 2 should be set up using BD CaliBRITE beads Cat No 349502 Instrument Setup with BD CaliBRITE Beads e Start up the instrument e Perform flow check e Prepare tubes of BD CaliBRITE beads e Launch BD FACSComp software e Run BD FACSComp using the lyse no wash procedure Note For detailed information on using BD FACSComp with BD CaliBRITE beads to set up the flow cytometer refer to the BD FACSComp Software User s Guide and the BD CaliBRITE Beads Package Insert Multi parameter Flow Cytometry Analysis of JC 1 As noted earlier there are a large number of factors that can affect JC 1 fluorescence emission both with respect to absolute intensity in the Green FL 1 and Red FL 2 channels and to the ratio of the two channels Figure 1 shows the results of 3 different experiments done on different days each analyzed using the instrument setup described in the section Flow Cytometer Setup Each experiment consists of a healthy control untreated population and a population induced to undergo apoptosis Major populations R1 are identified as FL 1 bright FL 2 bright in the healthy controls indicative of polarized Ay Minor populations are identified that have decreased fluorescence i
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