User Manual
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1. of 1X Wash Buffer or PBS If the cells are to be analyzed by a fluorescence plate reader the negative control and test samples should all be adjusted to approximately the same concentration of cells per mL The cells are now ready for analysis If the cells cannot be analyzed immediately they may be fixed by making a 1 10 v v dilution of the 10X Fixative into the cell suspensions from step 11 and mix The fixed cells may be stored at 2 to 8 C protected from light for up to 24 hours B Bridge International Inc 4 www b bridge com FLISP Assay Kit User Manual 13 For direct analysis of the FLISP reagent probed adherent monolayer carefully remove the overlay medium and discard 14 Add 1 0 to 2 0 mL of 1X Wash Buffer to the monolayer and allow the 1X wash Buffer to sit over the monolayer for 3 to 5 minutes to remove any unbound FLISP reagent 15 Wash the adherent monolayer two more times by repeating steps 13 and 14 Care must be taken to minimize the number of adherent cells displaced during the washing process 16 Following the final wash mount a cover slip with cells facing down onto a microscope slide containing a drop of 1X Wash Buffer or remove the plastic frame of the chamber slide and add a drop of 1X Wash Buffer and cover with a cover slip The adherent monolayer cells are now ready for analysis 17 If the adherent monolayer cells cannot be analyzed immediately they may be fixed by making a 1 10 v v dilution of the 10X Fixative i2. 75 30 74 91 2 51 M2 13 50 13 43 31 46 Negative control Non stimulated FLISP reagent stained cells FL 1 histogram Figure 5 Data 016 M1 100 10 10 102 104 FL1 Height Sample ID TL 3 POS Acquisition Date 18 May 04 Gate G1 Gated Events 9952 Total Events 10000 X Parameter FL1 Height Log Marker Gated Tota Mean All 100 00 98 70 29 74 M1 30 10 29 71 2 84 M2 56 28 55 55 50 44 Positive control stimulated FLISP reagent stained cells FL 1 histogram B Bridge International Inc 7 www b bridge com References 20 090 rM Ov tA CES O3 POS Johnson et al Leukemia 2000 14 1695 1703 Yousef et al J Biol Chem 2001 276 53 61 Chen et al J Biol Chem 2001 276 21434 42 Takayama et al Biochemistry 2001 40 1679 87 Magee et al Cancer Res 2001 61 5692 6 Ulutin amp Pak Radiat Med 2000 18 273 6 Yousef et al Genomics 2000 69 331 41 Lang et al Br J Cancer 2001 84 237 43 Zapata et al J Biol Chem 1998 273 6916 6920 Wright et al Biochem Biophys Res Commun 1998 245 797 803 Shi et al J Exp Med 1992 176 1521 9 Kam et al Biochim Biophys Acta 2000 1477 307 23 Jans et al J Cell Sci 1998 111 2645 54 Estabanez Perpina et al J Biol Chem 2000 321 1203 1214 Gorczyca et al Int J Oncol 1992 1 639 648 Bruno et al Leukemia 1992 6 1113 1120 Bruno et al Oncol Res 1992 4 29 35 Grabarek et al nt J Oncology 2002 20 225 2
3. 33 Grabarek et al Cell Cycle 2002 1 124 131 Bedner et al Exp Cell Res 2000 259 308 313 Smolewski et al Cytometry 2001 44 73 82 Darzynkiewicz et al Methods Mol Biol 2002 203 289 299 Smolewski et al Int J Oncol 2001 19 657 663 Amstad et al BioTechniques 2001 31 608 616 B Bridge International Inc 8 FLISP Assay Kit User Manual www b bridge com
4. B Bridge International Inc d FLISP Assay Kits User Manual RX 205 and RX 206 FOR RESEARCH USE ONLY Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc 2002 B Bridge International Inc All Rights Reserved 092 704 FLISP Assay Kit User Manual INTRODUCTION Serine proteases are defined by the presence of a serine residue at the active center of the enzyme which participates in the formation of an intermediate ester to transiently form an acyl enzyme complex Activated serine proteases play major roles in several different functions including Apoptosis 1 Markers of tumor malignancy 2 5 Diagnostic and prognostic indicators of breast carcinomas 6 7 and neck and head carcinomas 8 and Activities are also altered in a variety of other cell mediated diseases related to transplant rejection and infections 9 14 The most characterized enzymes of this type are trypsin and chymotrypsin Involvement of serine proteases in apoptosis has been mostly studied by observing whether particular apoptotic events can be prevented by the specific inhibitors of these enzymes Fragmentation of DNA in HL 60 cells treated with DNA topoisomerase inhibitors to induce a
5. bandpass filter with an excitation at 590 nm and an emission greater than 610 nm Cells with active serine protease activity will appear red when using the Sulforhodamine 101 labeled FLISP reagent e Hoechst stain can be observed by using a UV filter with excitation at 365 nm and an emission at 480 nm f Propidium Iodide PI can be observed using a broad bandpass filter with an excitation at 490 nm and an emission greater than 610 nm optimal settings would be an excitation at 535 nm and emission at 617 nm Figure 1 T Data 009 SS rmm T Tiny T Trinny T Tranny 00 10 10 10 104 FSC H Negative control Non stimulated FLISP reagent stained cells Data 009 Figure 2 FL2 height hiid T 103 104 102 FL1 Height Negative control Non stimulated FLISP reagent stained cells the cells in the lower left quadrant Figure 3 Data 016 FL2 Height 0 1 108 104 FL1 Height FW vs SS dot plot Fl 1 vs Fl 2 dot plot Readjust voltage gains to place Positive control stimulated FLISP reagent stained cells Fl 1 vs Fl 2 dot plot B Bridge International Inc 6 www b bridge com FLISP Assay Kit User Manual Figure 4 Data 009 M1 a 100 10 10 102 10 Sample ID TL 3 NEG Acquisition Date 18 May 04 Gate G1 Gated Events 10026 Total Events 10000 X Parameter FL1 Height Log Marker 96 Gated Tota Mean All 100 00 99 48 6 52 M1
6. ension Cell Procedure 1 Cultivate cells under optimal growth conditions to a concentration where cells remain healthy and do not exhibit signs of over growth or natural induction of apoptosis for most cell lines the cell concentrations will be less than 1 x 10 cells per mL 2 Splitcells into two or more cultures with one culture serving as a negative control no treatment or apoptosis induction and the other cultures as the test samples cells receiving treatment or apoptosis induction 3 Treat test samples with your reagent or induce apoptosis Continue to culture negative control and test samples for the same length of time under the same conditions as determined by your treatment 4 Following treatment count cells Cell concentrations between 0 5 x 10 to 1 x 10 cells per mL are optimal for incubation with the FLISP reagent For lower initial cell counts cells may be concentrated by centrifugation at 400 x g for 5 minutes then resuspend cells in an appropriate amount of cell media to achieve the desired concentration 5 Remove 0 490 mL 490 uL of each cell suspension and place into sterile tubes To each tube add 0 010 mL 10 uL of 50X FLISP reagent the 50X FLISP reagent is always diluted 1 50 in the cell suspension this gives a final concentration of 20 uM FLISP reagent This is a suggested starting concentration for the FLISP reagent optimal FLISP reagent concentration may be as low as 5 uM The incubation time and FLISP c
7. eter can calculate percentages of FLISP positive cells Fig 5 2 Fluorescence Plate Reader a Use black welled plates only b Fora 96 well plate transfer 0 050 mL 50 uL to 0 300 mL 300 uL of suspended cells per well Once the volume has been optimized deliver equal volumes to all wells for each experiment Setfluorescence plate reader to endpoint read d To read cells treated with FAM labeled FLISP reagent set excitation to 488 nm and emission to 530 nm with a 515 nm cut off filter e To read cells treated with Sulforhodamine 101 labeled FLISP reagent set excitation to 590 nm and emission to 620 nm with a 610 nm cut off filter 3 Fluorescence Microscopy a To view suspension cells place one drop of the cell suspension onto a microscope slide and cover with a cover slip b To view adherent cells mount a cover slip with cells facing down onto a microscope slide containing a drop of 1X Wash Buffer or remove the plastic frame of the chamber slide and add a drop of 1X Wash Buffer and cover with a cover slip Observe cells treated with FAM labeled FLISP reagent using a broad bandpass filter with an excitation at 490 nm and an emission greater than 520 nm Cells with active serine protease activity will appear green when using the FAM labeled FLISP reagent B Bridge International Inc 5 www b bridge com FLISP Assay Kit User Manual d Observe cells treated with Sulforhodamine 101 labeled FLISP reagent using a broad
8. m the cells and the results are read using flow cytometry fluorescence microscopy or fluorescence plate readers The reagents are used at low concentrations and normally do not have any adverse effects on the cells The FLISP products can also be used with ICT s caspase detection FLICA kits allowing the researcher to monitor serine protease and caspase activites simultaneously 18 19 The FLICA kits employ the use of fluorochrome labeled analogs of fluoromethyl ketone inhibitors of caspase activity 20 24 REAGENT PREPARATION Preparation of FLISP Serine Protease reagent The FLISP reagent is supplied as a concentrated lyophilized powder Unopened vials may be stored at 20 C for up to 18 months The reagent should be reconstituted just prior to use or freezing 1 Preparation of 250X FLISP reagent stock Dissolve lyophilized FLISP reagent in 0 050 mL 50 uL of DMSO Mix thoroughly washing the base and lower sides of the vial to ensure complete solubilization of the FLISP reagent DMSO reconstituted vials of FLISP reagent may be stored for up to 1 year at 80 C 2 Preparation of 50X FLISP reagent Add 0 2 mL 200 uL of PBS pH 7 4 to the 250X stock solution of FLISP reagent Once diluted in PBS the FLISP reagent must be used as soon as possible the 50X FLISP reagent is ready to add to the cells i e 0 010 mL 10 uL of 50X FLISP reagent added to 0 490 mL 490 uL of cells 3 FAM labeled FLISP reagents require a 488 10 nm e
9. nto 1X Wash Buffer and add one drop of this to the cell surface followed by a cover slip The fixed slides may be stored at 2 to 8 C protected from light for up to 24 hours Do not fix cells if they are to be counterstained with PI or Hoechst stain ANALYSIS 1 Flow Cytometry a Setup acquisition template dot plots SS vs FW Fig 1 FL 1 vs FL 2 Fig 2 and FL 1 histogram Figs 4 and 5 b Open compensation and voltage gain controls Although the FLISP products are analyzed on a histogram FL 1 vs FL 2 dot plot should be used to properly setup interment i Run the negative control non stimulated FLISP reagent stained cells and set up FW vs SS by drawing a gate around target cell population Fig 1 ii Gating on the target cell population setup the Fl 1 vs Fl 2 dot plot Readjust voltage gains toplacethe cells in the lower left quadrant Fig 2 iii Run the positive control stimulated FLISP reagent stained cells and check to confirm that the population of FLISP labeled cells have moved to the right Fig 3 If the cells move up or down the Y axis adjust the compensation iv If compensation was adjusted in the previous step run the negative control again to ensure the proper setup v Running the negative control setup a histogram on the FL 1 channel Make sure that the peak is to the right of the Y axis and within the first decade set markers as demonstrated in figure 4 vi By setting the marker as in step 1 b v the flow cytom
10. oncentration should be optimized for cell type and experimental conditions 6 Incubate the cells with the FLISP reagent between 30 minutes and 2 hours at 37 C periodically resuspending the cells every 20 to 30 minutes Incubation time will vary depending on cell count and cell type During B Bridge International Inc 3 www b bridge com 11 FLISP Assay Kit User Manual the incubation period the FLISP reagent enters the cell and binds irreversibly to the catalytic site of the active serine proteases a Ifcells are to be stained with Hoechst stain add 2 5 uL 0 596 v v of the Hoechst solution at the end of the FLISP incubation step and incubate for an additional 5 minutes After incubation with the FLISP reagent gently centrifuge the cells at less than 400 x g for 5 minutes to pellet the cells Carefully pull off the supernatant and discard it Resuspend the cell pellet in 1 0 to 2 0 mL of 1X Wash Buffer to remove any unbound FLISP reagent Wash the cells two more times by repeating steps 7 and 8 Resuspend the final washed cell pellet in 0 5 mL 500 uL of 1X Wash Buffer or PBS If the cells are to be analyzed by a fluorescence plate reader the negative control and test samples should all be adjusted to approximately the same concentration of cells per mL The cells are now ready for analysis a If cells are to be stained with Propidium Iodide add 2 5 uL 0 5 v v of the PI solution to the resuspended cells If the cells cann
11. ot be analyzed immediately they may be fixed by making a 1 10 v v dilution of the 10X Fixative into the cell suspensions from step 10 and mix The fixed cells may be stored at 2 to 8 C protected from light for up to 24 hours Do not fix cells if they are to be counterstained with PI or Hoechst stain Adherent Cell Procedure 1 10 11 12 Cultivate cells under optimal growth conditions to a concentration where cells are not becoming overgrown and beginning to slough off of the flask surface When cells are overcrowded the level of apoptotic cells tends to increase Trypsinize healthy growing cells and transfer to cell culture flasks slides or chambers Cells should be seeded at a sufficient number to provide an initial coverage of 3096 to 5096 confluency Include at least one flask slide or chamber as a negative control that will receive no treatment When the cells have reached a level between 60 and 80 confluency they can be treated with your reagent or induced into apoptosis Continue to culture negative control and test samples for the same length of time under the same conditions as determined by your treatment To each monlayer of cells add an amount of the 50X FLISP Reagent to make a 1 50 dilution in the cell media i e if 0 490 mL 490 uL of cell media is used to culture the cells add 0 010 mL 10 uL of 50X FLISP reagent The final concentration of FLISP reagent will be 20 uM This is a suggested starting concentration for
12. poptosis was prevented by the use of an irreversible serine protease inhibitor such as N tosyl L phenylalanine chloromethyl ketone TPCK which inhibits chymotrypsin 15 The same inhibitor also inhibited nuclear fragmentation as well as fragmentation of DNA in other cell types including thymocytes treated with the corticosteroid prednisolone 16 17 It is now possible to measure serine protease activity in whole living cells using the FLISP Fluorescent Labeled Inhibitors of Serine Proteases line of products 18 19 These patent pending products are fluorochrome labeled analogs of serine protease inhibitors labeled with either Carboxyfluorescein FAM or Sulforhodamine 101 also known as Texas Red Current products include FAM Phenylalanine chloromethyl ketone FFCK SR 101 phenylalanine chloromethyl ketone SFCK FAM Leucine chloromethyl ketone FLCK and SR101 Leucine chloromethyl ketone SLCK The FAM labeled products are also available with a spacer FSFCK and FSLCK Note the FFCK FSFCK and SFCK terminology represents the current nomenclature of F for phenylalanine as opposed to P which was used when the TPCK inhibitor was originally described The FLISP reagents are cell permeable and can be used to detect serine proteases in suspension cells and adherent cells Once they enter the cell they irreversibly bind to the active catalytic site of the respective active serine protease The unbound reagent is washed fro
13. the FLISP reagent optimal FLISP reagent concentration may be as low as 5 uM The incubation time and FLISP concentration should be optimized for cell type and experimental conditions Incubate the cells with the FLISP reagent between 30 minutes and 2 hours at 37 C The overlay medium containing the FLISP reagent should be agitated periodically every 20 to 30 minutes to ensure an even labeling process a Ifcells are to be stained with Hoechst stain add 2 5 uL 0 596 v v of the Hoechst solution at the end of the FLISP incubation step and incubate for an additional 5 minutes After FLISP labeling the adherent cells can be processed by two different methods for analysis The adherent monolayer can be analyzed directly go to step 13 or the cells can be removed prior to analysis beginning with step 7 Carefully aspirate the cell supernatant from the monolayer and place into a sterile centrifuge tube Trypsinize the remaining cells off of the monolayer surface and combine with the cells from the original supernatant in cell culture media containing fetal bovine serum to inactivate the trypsin enzymatic activity Gently centrifuge the cells at less than 400 x g for 5 minutes to pellet the cells Carefully pull off the supernatant and discard it Resuspend the cell pellet in 1 0 to 2 0 mL of 1X Wash Buffer to remove any unbound FLISP reagent Wash the cells two more times by repeating steps 8 and 9 Resuspend the final washed cell pellet in 0 5 mL 500 uL
14. urs 3 Do not attempt to fix cells that will be stained with Propidium Iodide or Hoechst Stain Propidium Iodide Propidium Iodide PI is provided ready to use at a concentration of 250 ug mL PI stains necrotic dead and membrane compromised cells and can be used to distinguish between live cells and dead cells 1 Propidium Iodide can be used with FAM labeled FLISP reagent for bi color analysis 2 The PI solution is added to the suspended cells at a 0 596 v v amount In a typical reaction mixture 2 5 uL of PI is added to 0 500 mL 500 uL of suspended cells or overlay medium for adherent monolayer cells 3 PI has a broad excitation range 488 to 492 nm will provide enough excitation the optimal excitation is 535 nm and has an emission maximum at greater than 610 nm peak emission is 617 nm Hoechst Stain Hoechst stain is provided ready to use at a concentration of 200 ug mL It can be used to label the nuclei of dying cells after labeling with the FLISP reagent 1 Hoechst stain can be used with FAM or sulforhodamine 101 labeled FLISP reagent for bi color analysis 2 The Hoechst solution is added to the suspended cells or adherent monolayer at a 0 5 v v amount Ina typical reaction mixture 2 5 uL of Hoechst solution is added to 0 500 mL 500 uL of suspended cells or overlay medium for adherent monolayer cells 3 Hoechst stain is detected using a UV filter with excitation at 365 nm and an emission at 480 nm CELL PREPARATION Susp
15. xcitation and gt 520 nm emission optics pairing 4 Sulforhodamine 101 labeled FLISP reagents require a 590 10 nm excitation and gt 610 nm emission optics pairing Preparation of 1X Wash Buffer The wash buffer comes as a 10X concentrate which must be diluted to 1X with DI H20 prior to use 1 If necessary gently warm the 10X concentrate to completely dissolve any salt crystals that may have formed during storage B Bridge International Inc 9 www b bridge com FLISP Assay Kit User Manual 2 For the 25 test FLISP kit Add the entire contents of the 10X wash buffer 15 mL to 135 mL of DI H20 making a total of 150 mL of 1X wash buffer 3 For the 100 test FLISP kit Add the entire contents of the 10X wash buffer 60 mL to 540 mL of DI H20 making a total of 600 mL of 1X wash buffer 4 1X wash buffer may be stored up to 30 days at 2 to 8 C If any salt crystals form warm to room temperature and stir 5 minutes until all crystals have dissolved 10X Fixative The fixative comes as a 10X concentration of a formaldehyde solution designed to cross link cell components and will not interfere with the carboxyfluorescein or sulforhodamine 101 labeling once the FLISP reaction has taken place 1 After FLISP labeling add the 10X Fixative to the cell solution at a volume that will give a 1 10 dilution To 0 450 mL 450 uL of cells add 0 050 mL 50 uL of 10X Fixative 2 Fixed cells may be stored on ice or at 2 to 8 C for up to 24 ho
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