Technical Bulletin. Multiple Methods for Detecting Apoptosis on the BD Accuri C6 Flow Cytometer. Introduction
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1 March 212 Multiple Methods for Detecting Apoptosis on the BD Accuri C6 Flow Cytometer Contents 1 Introduction 2 Annexin V 4 JC-1 5 Caspase-3 6 APO-BrdU and APO-Direct Introduction Apoptosis (programmed cell death) is an important biological process for both development and normal tissue homeostasis. Dysregulation of apoptotic pathways can lead to disease. Methods for detecting apoptosis include Western blot, immunofluorescence, enzymatic assays, and flow cytometry. Flow cytometry is especially powerful because researchers can gain quantitative data on both apoptotic and dead cells within whole populations and cell subsets. The BD Accuri C6 personal flow cytometer is well suited to the study of apoptosis. With the ability to detect four fluorochromes in addition to forward and side scatter, the instrument can perform most flow cytometric apoptosis assays, including Annexin V, caspase activation, PARP cleavage, mitochondrial change, and DNA fragmentation. Powerful yet easy to use, the BD Accuri C6 allows both new and experienced flow cytometry users to perform these established assays. This technical bulletin presents examples of several popular BD Biosciences apoptosis kits (Table 1) run on the BD Accuri C6. It discusses the background of the assays and includes suggestions to optimize results.
2 March 212 Table 1. Selected methods for detecting apoptosis using flow cytometry. Apoptosis Indicator Assay Examples Cat. No. Plasma membrane alterations (Phosphatidylserine exposure) Annexin V binding assays Single conjugates Annexin V kits Annexin V FITC Apoptosis Detection Kit Annexin V PE Apoptosis Detection Kit Mitochondrial changes JC-1 assays BD MitoScreen (JC-1) Kit Caspase activation DNA fragmentation Caspase activity assay kits and reagents TUNEL assays Caspase-3 Active Form PE Apoptosis Kit APO-BrdU Apoptosis Detection Kit APO-Direct Apoptosis Detection Kit Annexin V Changes in the plasma membrane are one of the first detectable characteristics of the apoptotic process. In normal cells, phosphatidylserine (PS) molecules are confined to the inner leaflet of the plasma membrane (Figure 1). During apoptosis, these molecules externalize and can be bound to labeled Annexin V (FITC or PE) in the presence of calcium. Dead cells can be excluded with membrane-impermeant dyes such as propidium iodide (PI) or 7-AAD. Annexin V-PE Conjugate Plasma Membrane Phospholipid Flipping Ca ++ Ca ++ Ca ++ Apoptosis Normal Cell Cytoplasm Externalization of Phosphatidylserine Apoptotic Cell Cytoplasm Figure 1. Changes in the plasma membrane are an early sign of apoptosis.
3 March 212 Page 3 Figure 2. Flow cytometric analysis of FITC Annexin V staining. Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with 6 µm of camptothecin or.1% DMSO (negative control) for 4 hours to induce apoptosis. Cells were stained with FITC Annexin V and PI according to the BD Pharmingen Annexin V FITC Apoptosis Detection Kit staining protocol (Cat. No ). Results: Camptothecin treatment (lower plots) resulted in an increase in early apoptotic cells (PI Annexin V +, shown in green) compared to the DMSO control (upper plots). Dead cell (PI +, red) and live cell (PI Annexin V, black) populations were easily distinguished. Data was acquired on a BD Accuri C6 flow cytometer using BD Accuri C6 software. 5, 1,, 1,74,234 DMSO FITC + PI 87.4% 199,729 5,, 1,58, FL3 Propidium Iodide-A DMSO FITC + PI Q2-UL.1% Q2-LL 86.% Q2-UR 11.1% Q2-LR 2.8% FL1 Annexin V FITC-A Campto FITC + PI Campto FITC + PI 5, 1,, 1,74, % 199,729 5,, 1,58, FL3 Propidium Iodide-A Q2-UL.% Q2-LL 63.2% Q2-UR 9.1% Q2-LR 27.7% FL1 Annexin V FITC-A Figure 3. Flow cytometric analysis of PE Annexin V staining. Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with 6 µm of camptothecin or.1% DMSO (negative control) for 4 hours to induce apoptosis. Cells were stained with PE Annexin V and 7-AAD according to the BD Pharmingen Annexin V PE Apoptosis Detection Kit staining protocol (Cat. No ). Results: Camptothecin treatment (lower plots) resulted in an increase in early apoptotic cells (7-AAD Annexin V +, shown in orange), compared to the DMSO control (upper plots). Dead cell (7-AAD +, red) and live cell (7-AAD Annexin V, black) populations were easily distinguished. Data was acquired on a BD Accuri C6 flow cytometer using BD Accuri C6 software. 1,634,686 1,, 5, 1,634,686 1,, 5, DMSO PE + 7AAD 88.1% Campto 6 um PE + 7AAD 48.5% 5,, 8,66,171 5,, 8,66,171 FL3 7-AAD-A FL3 7-AAD-A DMSO PE + 7AAD Q4-UL.5% Q4-LL 88.4% 1 3 Q4-UL.2% Q4-LL 64.4% 1 4 FL2 Annexin V PE-A Campto 6 um PE + 7AAD FL2 Annexin V PE-A Q4-UR 8.5% Q4-LR 2.6% Q4-UR 9.3% Q4-LR 26.1% 1 6.3
4 March 212 JC-1 Changes in mitochondrial membrane potential (ΔΨ m ) are another early marker for apoptosis. Lypophilic cationic fluorochromes such as JC-1 penetrate cells. In healthy cells, JC-1 accumulates in the mitochondria and forms aggregates. In apoptotic cells, however, JC-1 does not accumulate in the mitochondria and remains in the cytoplasm as monomers. Because monomers and aggregates of JC-1 have different emission spectra, changes in ΔΨ m can be determined by comparing the ratio of fluorescence between the FL1 and FL2 channels. 2,, 5,93,85 A2 K562 untreated JC-1 9.% 5,, 1,, 14,54,969 JC-1 FL-2-A A2 K562 untreated JC-1 P9 87.6% 1.6% JC-1 FL-1-A JC-1 FL-2-A A3 K562 CCCP JC-1 P9 12.9% 84.% JC-1 FL-1-A Figure 4. Flow cytometric analysis of BD MitoScreen staining. K562 cells (human chronic myelogenous leukemia; ATCC CCL-243) were treated with 1 µm of CCCP (in DMSO) for 5 minutes at 37ºC to induce apoptosis. The cells were stained with JC-1 (1:2,5 dilution in assay buffer) for 15 minutes at 37ºC, according to the BD MitoScreen protocol (Cat. No ). The cells were washed with assay buffer as described in the kit insert and collected on the BD Accuri C6 for 3 seconds on fast speed. Results: CCCP treatment resulted in a shift in mitochondrial membrane potential (red to green). Data was acquired on a BD Accuri C6 flow cytometer using BD Accuri C6 software.
5 March 212 Page 5 Caspase-3 Changes in ΔΨ m as a result of apoptotic triggers make the mitochondrial membrane more permeable and release soluble proteins such as cytochrome c and procaspases. Procaspases are activated by protein cleavage and, in turn, cleave other proteins. This leads to a loss of cellular function and structure. Several caspases are important for apoptosis, including caspase-3, -8, and -9. Methods of detecting caspase cleavage include fluorogenic substrates as well as antibodies specific to the cleaved (activated) forms of caspases. 1,22,61 5, 1,22,61 5, DMSO camptothecin 95.7% 8.3% 2,, 2,, 5,218,844 5, 1,131,797 5, 1,131, ,218, DMSO 1 3 PE active caspase-3-a camptothecin PE active caspase-3-a Count PE active caspase-3-a 8 6 Count 4 2 DMSO V2-L 97.% camptothecin V2-L 74.4% V2-R 3.% V2-R 25.6% PE active caspase-3-a Figure 5. Flow cytometric analysis of active Caspase-3 staining. Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with 6 µm of camptothecin or.1% DMSO (negative control) for 4 hours to induce apoptosis. Cells were permeabilized, fixed, and stained according to the BD Pharmingen Caspase-3 Assay Kit staining protocol (Cat. No ). Results: Camptothecin treatment (lower plots) resulted in an increase in active Caspase-3 expression (red) compared to the DMSO control (upper plots), which was primarily negative (green). Data was acquired on a BD Accuri C6 flow cytometer using BD Accuri C6 software.
6 March 212 APO-BrdU and APO-Direct DNA fragmentation is one of the final stages of apoptosis. BD Biosciences provides two kits to detect DNA fragmentation using flow cytometry. The BD APO-BrdU Kit uses a reaction catalyzed by terminal deoxynucleotidyl transferase (TdT) to detect fragmentation. The method is often called end labeling or TUNEL (TdT dutp nick end labeling). In this assay, TdT catalyzes a templateindependent addition of bromolated deoxyuridine triphosphates (Br-dUTP) to the 3'-hydroxyl (OH) termini of double- and single-stranded DNA. After the Br-dUTP is incorporated, the cells are stained with labeled anti-brdu, and the DNA terminal sites are identified using flow cytometry. TdT + Br-dUTP FITC-Labeled Anti-BrdU mab DNA Strand Breaks Add Br-dUPT to 3 - OH DNA Ends Antibody Labeled Break Sites Figure 6. Schematic representation of APO-BrdU labeling. The enzyme TdT catalyzes a template-independent addition of Br-dUTP to the 3'-hydroxyl ends of double- and single-stranded DNA. After Br-dUTP incorporation, DNA break sites are identified by a FITC-labeled anti-brdu monoclonal antibody. The BD APO-Direct Kit uses the same catalyst (TdT) in a single-step method that labels DNA breaks with a dutp antibody. TdT + FITC-dUTP DNA Strand Breaks FITC Labeled Break Sites Figure 7. Schematic representation of APO-Direct labeling. The enzyme TdT catalyzes a template-independent addition of FITC-labeled deoxyuridine triphosphates (FITC-dUTP) to the 3'-hydroxyl ends of double- and single-stranded DNA. When the FITC-labeled dutps are incorporated, the DNA break sites can be identified.
7 March 212 Page 7 Figure 8. Flow cytometric analysis of APO-BrdU staining. Positive control cells (human lymphoma cell line stimulated to undergo apoptosis, bottom row) and negative control cells (untreated lymphoma cell line, top row), both included in the BD APO-BrdU Kit (Cat. No ), were stained according to the kit insert. Samples were collected for 3 seconds on fast speed on a BD Accuri C6 flow cytometer, and data was acquired using BD Accuri C6 software. Clumped cells were excluded by gating on the DNA Area vs Height plot (middle plots). Results: The positive control cells showed a significant increase in apoptotic cells (R1) compared to the negative control. 2,, 4,, 5,392,677 2,, 4,, 5,392,677 D4 - control BRDU 58.6% 5,, 13,182,99 D5 + control BRDU 39.1% 5,, 13,182,99 FL-2 DNA-H 2, 4, 633,86 FL-2 DNA-H 2, 4, 633,86 D4 - control BRDU 87.% 5, 936,562 FL-2 DNA -A D5 + control BRDU 68.4% 5, 936,562 FL-1 FITC BrdU-A FL-1 FITC BrdU-A D4 - control BRDU ate: ( in all) R1 2.7% 1 2, 4, 6, 777,654 FL-2 DNA -A D5 + control BRDU ate: ( in all) R1 7.% 1 2, 4, 6, 777,654 Figure 9. Flow cytometric analysis of APO-Direct staining. Positive control cells (human lymphoma cell line stimulated to undergo apoptosis, bottom row) and negative control cells (untreated lymphoma cell line, bottom row), both included in the BD APO-Direct Kit (Cat. No ), were stained according to the kit insert. Samples were collected for 3 seconds on fast speed on a BD Accuri C6 flow cytometer, and data was acquired using BD Accuri C6 software. Clumped cells were excluded by gating on the DNA Area vs Height plot (middle plots). Results: The positive control cells showed a significant increase in apoptotic cells (R1) compared to the negative control. 2,, 4,, 5,392,677 2,, 4,, 5,392,677 A4 Kit neg control Direct 66.2% 5,, 13,182,99 A5 Kit pos control Direct 63.% 5,, 13,182,99 FL-2 DNA-H 2, 4, 633,86 FL-2 DNA-H 2, 4, 633,86 A4 Kit neg control Direct 82.9% 5, 936,562 A5 Kit pos control Direct 81.1% 5, 936,562 FL-1 FITC dutp-a FL-1 FITC dutp-a A4 Kit neg control Direct ate: ( in all) R1 1.2% 1 2, 4, 6, 777,654 A5 Kit pos control Direct ate: ( in all) R1 54.4% 1 2, 4, 6, 777,654
8 March 212 For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. 212 BD BD Biosciences 235 Qume Drive San Jose, CA US Orders: BD Accuri Technical Support: bdbiosciences.com
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