For research use only In situ detection kit for programmed Cell Death MEBSTAIN Apoptosis Kit Direct CODE No. 8445
In situ detection kit for Programmed Cell Death MEBSTAIN Apoptosis Kit Direct Cat. No. 8445 Intended Use The MEBSTAIN Apoptosis Kit Direct is intended for the detection of apoptotic cells in research samples. It is suitable for histochemistry, cytospin preparation, and flow cytometry. For research use only. Not for use in diagnostic procedures. Summary and Explanation Programmed cell death is a selective process of physiological cell deletion. It plays a major role in developmental biology and in maintenance of homeostasis in vertebrates (1-4). For example, during maturation, T cells recognizing self antigens are destroyed by programmed cell death (5). Programmed cell death is morphologically known as apoptosis. Apoptosis is accompanied by condensation of cytoplasm, loss of plasma membrane microvilli, condensation and fragmentation of nuclei, and extensive degradation of chromosomal DNA into oligomers of about 180 bp. Fragmentation of nuclear DNA is a biochemical hallmark of apoptosis. The TdT-mediated dutp-biotin nick end labeling method (TUNEL method) developed by Gavrieli et al (6) has enabled in situ visualization of DNA fragmentation at the single cell level and is a more sensitive method than conventional morphological techniques(7). The MEBSTAIN Apoptosis Kit Direct is an apoptosis detection kit based on the TUNEL method. The main advantage of this rapid and simple procedure is the use of fluorescein-dutp to label DNA strand breaks. This allows the direct detection of DNA fragmentation. 1
Principle In cells in which apoptosis occurs, the chromatin DNA is cut by endonuclease at linker DNA site between nucleosomes. This results in DNA fragments which are multimers of the nucleosomal units. In the TUNEL method, 3'-OH DNA ends generated by DNA fragmentation is nick end-labeled with fluorescein-dutp, mediated by terminal deoxynucleotidyl transferase (TdT). This method allows specific staining. Materials Supplied Each kit contains: Materials Proteinase K (PK) TdT TdT buffer II FITC-dUTP TB buffer (x10) Quantity 04.ml x 1vial 0.1ml x 1vial 2ml x 3vials 0.1ml x 1vial 50ml x 1vial 2
Precautions 1. This kit is for research use only. Not for use in diagnostic procedures. 2. TdT and TdT buffer II contain 200mM cacodylic acid. As cacodylic acid is an arsenic compound, do not dispose materials containing TdT and TdT buffer II into a drain. 3. During procedure, do not allow tissue sections to become dry. This will cause erroneous results. Perform procedures in a humidified chamber to avoid drying of tissue sections. Storage and Stability All kit components must be stored at -20ºC. All reagents are stable until the expiration date when stored at -20ºC. After thawing TB buffer (x10), it should be stored at 4ºC. Assay Procedure Protocol for Immunohistochemistry Materials required but not provided Xylene Ethanol (100%, 90%, 80%) PBS Mounting medium (90% glycerin, 10%PBS) Cover slip Distilled water Moist chamber Propidium Iodide (0.5 µg/ml) *It is for counterstaining. Buffered paraformaldehyde Preparation of reagents a) Proteinase K (PK) solution (not required for frozen tissue sections) Just prior to use, prepare PK solution by diluting PK 1: 24 (v/v) with PBS. 3
Warm PK solution to 37ºC. The remainder of Proteinase K should be stored at -20ºC. b) TdT solution Just prior to use, prepare TdT solution (preparation should be done in the ice) mix TdT buffer II, FITC-dUTP, and TdT at 18: 1: 1. Prepare multiples of 50 µl as follows: 45 µl TdT buffer II 2.5 µl FITC-dUTP 2.5 µl TdT 50 µl Total The remainder of TdT buffer II, FITC-dUTP and TdT should be stored at -20ºC. c) TB solution Just prior to use, dilute TB buffer (x10) 1:9 with distilled water for a Coplin jar. After thawing TB buffer (x10), it should be stored at 4ºC. d) Buffered paraformaldehyde (fixation reagent) Prepare 4% paraformaldehyde in 0.1M NaH 2PO 4, ph7.4 (4%PFA) e) Propidium Iodide (for counterstaining) Prepare Propidium Iodide by diluting with PBS to obtain a concentration of 0.5 µg/ml. Protocol 1. Preparation of tissue sections Use glass slides pretreated with silane. 4% paraformaldehyde in 0.1M NaH 2PO 4, ph7.4 (4%PFA) as recommended as a fixation regent for sensitive detection. Do not use ethanol and acetone because they may cause non-specific staining. 3 5 µm of sections are suitable for staining. a) Paraffin embedded tissue sections Fix tissue samples in 4% PFA and embed in paraffin. Proceed to step 2 b) Frozen tissue sections Post-fix cryosections in 4% PFA 4
IMPORTANT: After post-fixation of frozen tissue sections, wash slide with PBS for 5 min. 2 times. After this procedure, be careful not to allow tissue sections to dry. Proceed directly to step 4 (<<DNA nick end labeling>>) 2. Deparaffinization Deparaffinize tissue sections in a Coplin jar by the following procedure: Xylene 3min., 3times 100% EtOH 3min., 2times 90% EtOH 3min., 1time 80% EtOH 3min., 1time Washing 2min., 4times with distilled water After this procedure, be careful not to allow tissue sections to dry. 3. Treatment with proteinase K (PK) * * Note: this step is not required for frozen tissue sections. 1) Pipet 500 µl of PBS on the section and incubate for 30 min. at 37ºC. (Or put the slide into a jar filled with PBS which is warmed to 37ºC, and incubate 15-30 min. at 37ºC.) 2) After removing PBS buffer by tapping it off, pipet 250 µl of PK solution on the section and incubate for 30min. at 37ºC. 3) Wash slide with distilled water for 2 min., 4 times. In the meantime, prepare TdT solution. (Preparation should be done on ice.) 4. DNA nick end labeling 1) Pipet 50 µl of TdT buffer II on the section and incubate for 5-10 min. at room temperature. 2) After removing TdT buffer II by tapping it off, pipet 50 µl of TdT solution (mixture of 45 µl of TdT buffer II, 2.5 µl of FITC-dUTP, and 2.5 µl of TdT) and incubate for 60 min. at 37ºC. 5
3) After removing TdT solution by tapping it off, immerse the slide in TB solution in a Coplin jar and incubate for 15 min. at room temperature. 4) Wash slide with PBS for 5 min., 3 times. 5. Counterstaining (In case of need) 1) Pipet 50 µl of 0.5 µg/ml Propidium Iodide on the section and incubate for 15-20 min. at 4ºC. 2) Wash slide with PBS for 3 min. 6. Mounting and microscopy 1) Mount with mounting medium (90% glycerin, 10%PBS) under coverslip. 2) View by fluorescent microscopy. Protocol for FLOW CYTOMETRY Materials required but not provided PBS PBS containing 0.2% BSA Buffered paraformaldehyde 70% Ethanol Preparation of regents a) TdT solution Just prior to use, prepare TdT solution (preparation should be done in the ice). 1) Mix TdT buffer II, FITC-dUTP and TdT in the ratio of 18:1:1 (27 µl: 1.5 µl: 1.5 µl). Prepare multiples of 30 µl of TdT solution and store at 4ºC. 2) As negative control, mix and prepare TdT buffer II and FITC-dUTP in the ratio of 19:1 (28.5 µl: 1.5 µl). The remainder of TdT buffer II, FITC-dUTP and TdT should be stored at -20ºC. b) Buffered paraformaldehyde (fixation reagent) Prepare 4% paraformaldehyde in 0.1M NaH 2PO 4, ph7.4 (4%PFA). 6
Protocol 1. Fixation of cells Wash the cells with PBS containing 0.2% BSA several times. Then fix them with 4% PFA at 4ºC for 30 min., followed by washing 2 times with PBS containing 0.2% BSA. 2. Permeabilization Add 200 µl of 70% Ethanol to the cell pellet. Then incubate it for 30 min. at -20ºC to permeabilize. In the meantime, prepare TdT solution. (Preparation should be done on ice.) (If there is sufficient time before test, suspend it in 70% of Ethanol, store at -20ºC and test within one week.) 3. DNA nick end labeling After washing with PBS containing 0.2% BSA 2 times, add 30 µl of TdT reaction reagent to the cell pellet and incubate for 1 hour at 37ºC. 4. Wash 2 times with PBS containing 0.2% BSA, and suspend the cell pellet to 200-500 µl of PBS containing 0.2% BSA. Note 1) All assays should be performed with a positive control and negative control. Peripheral blood mononuclear cells or cultured cells (more than 95% viable) such as Raji cells, etc. are recommended to be used as a negative control and those above treated with DNase I (by 1 µg/ml DNase I, 1 hour 37ºC) are recommended to be used as a positive control. DNase I treatment should be done after permeabilization. Note 2) 4% PFA (in 0.lM NaH 2PO 4, ph7.4) is recommended to be used as a fixation reagent. Do not use ethanol and acetone because they may cause non-specific staining. 7
Protocol for CYTOSPIN SAMPLES Materials Required but not provided PBS containing 2% fetal calf serum (FCS) and 0.1%NaN 3 Distilled water containing 0.2% BSA Distilled water containing 0.5% Tween 20 and 0.2% BSA Buffered paraformaldehyde Propidium Iodide (0.5 µg/ml) * It is for counterstaining. Mounting medium (90% glycerin, 10% PBS) Cover slip Moist chamber Preparation of regents a) TdT solution Just prior to use, prepare TdT solution (preparation should be done on ice). 1) Mix TdT buffer II, FITC-dUTP and TdT in the ratio of 18:1:1 (45 µl: 2.5 µl: 2.5 µl). Prepare multiples of 50 µl of TdT solution and store at 4ºC. 2) As negative control, mix and prepare TdT buffer II and FITC-dUTP in the ratio of 19:1 (47.5 µl: 2.5 µl). The remainder of TdT buffer II, FITC-dUTP and TdT should be stored at -20ºC b) Buffered paraformaldehyde (fixation reagent) Prepare 4% paraformaldehyde in 0.1M NaH 2PO 4, ph7.4 (4%PFA). Protocol 1. Cytospin Wash the cell with PBS (2% FCS, 0.1%NaN 3) several times and followed by cytospin according to the normal method. Condition of cytospin is as follows (protocol may differ with the instrument used). Number of cell : 50 100 µl at the conc. of 10 5 /ml per slide Rotation : 300-500 x g Time : 5-10 min. 8
2. Fixation of cells Dry the cytospin slide for about 1 hour using a fan. Then, fix the cells at 4ºC for 15 min. by 4% PFA (in 0.1M NaH 2PO 4, ph7.4) 3. Permeabilization Shake off 4% PFA and add 0.5% Tween 20 (0.2% BSA in PBS) to the cells. Treat for about 15 min. at room temperature to permeabilize. In the meantime, prepare TdT solution and negative control. (Preparation should be done on ice.) 4. DNA nick end labeling Wash 3 times with distilled water, then add 50 µl of TdT solution to the cells. Incubate for 1 hour at 37ºC. Wash slide 3 times with PBS. 5. Counterstaining (In case of need) 1) Pipet 50 µl of 0.5 µg/ml Propidium Iodide on the cells and incubate for 15-20 min. at 4ºC. 2) Wash slide with PBS for 3 min. 6. Mounting and microscopy 1) Mount with mounting medium (90% glycerin, 10%PBS) under coverslip. 2) View by fluorescent microscopy. Note 1) Test is recommended to be done with positive control and negative control. Peripheral blood mononuclear cells or cultured cells (more than 95% viable) such as Raji cells, etc. are recommended to be used as a negative control and those above treated with DNase I (by 1 µg/ml DNase I, 1 hour 37ºC) are recommended to be used as a positive control. DNase I treatment should be done after permeabilization. Note 2) 4% PFA (in 0.1M NaH 2PO 4, ph7.4) is recommended to be used as a fixation reagent. Do not use ethanol or acetone because they may cause non-specific staining. 9
References 1) Wyllie, A. H. Apoptosis (The 1992 Franck Rose Memorial Lecture). 1993. Br. J. Cancer 67, 205-208 2) Steller, H. Mechanisms and genes of cellular suicide. 1995. Science 267, 1445-1449 3) Mori, C., Nakamura, N., Okamoto, Y., Osawa, M., Shiota, K. Cytochemical identification of programmed cell death in the fusing fetal mouse palate by specific labeling of DNA fragmentation. 1994. Anat. & Embryol. 190, 21-28. 4) Mori, C., Nakamura, N., Kimura, S., Irie, H., Takigawa, T., Shiota, K. Programmed Cell death in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation. 1995. Anat. Rec. 242, 103-110. 5) Krammer, P. H., Behrmann, I., Daniel, P., Dhein, J., Debatin, K.-M Regulation of apoptosis in the immune system. 1994. Curr. Opin. Immunol. 6, 279-289 6)Gavrieli, y., Sherman, Y., Ben-Sasson, S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. 1992. J. Cell Biol. 119, 493-501. 7) Carbonari, M., Cibati, M., Fiorilli. M. Measurement of apoptotic cell in peripheral blood. 1995. Cytometry 22, 161-167. 10 October 31, 2003