User Manual-ENZ-51035-K100 - ProteoStat Aggresome Detection
Contents
1. D rcc j m ECC rcc rcc j m LL occ ECL 7 Enzo Enabling Discovery in Life Science ProteoStat Aggresome Detection Kit for flow cytometry and fluorescence microscopy Instruction Manual Cat No ENZ 51035 K100 For research use only Rev 1 2 0 January 2011 Notice to Purchaser The ProteoStat Aggresome Detection Kit is a member of the CELLestial product line reagents and assay kits comprising fluorescent molecular probes that have been exten sively benchmarked for cell analysis applications CELLestial reagents and kits are optimal for use in demanding cell analysis applications involving confocal microscopy flow cytometry microplate readers and HCS HTS where consistency and reproducibility are required This product is manufactured and sold by ENZO LIFE SCIENCES INC for research use only by the end user in the research market and is not intended for diagnostic or therapeutic use Purchase does not include any right or license to use develop or otherwise exploit this product commercially Any commercial use development or exploitation of this product or development using this product without the express prior written authorization of ENZO LIFE SCIENCES INC is strictly prohibited Limited Warranty Th2. Results are presented by histogram overlays Control cells were stained as well but display low fluorescence In the samples treated with 5 uM MG 132 for 18 hours The ProteoStat aggresome red dye signal increases over 2 fold indicating that MG 132 induced aggresome formation in Jurkat cells The APF value is about 66 4004 1 Control 3004 Treatment Mean FL3 Signal Control 113 Count 2004 Treated 5 uM MG 132 335 1004 Aggresome Propensity Factor APF 66 FL3 H Figure 5 Flow cytometry based cell aggresome analysis Jurkat cells were mock induced with 0 2 DMSO or induced with 5 uM MG 132 for overnight hours at 37 C After treatment cells were fixed and incubated with ProteoStat aggresome dye then analyzed by flow cytometry without washing using a 488 nm laser with fluorescence detection in the FL3 channel Results are presented as histogram overlays In MG 132 treated cells fluorescent green signal increases over 2 fold The described assay allows assessment of the effects of protein aggregation Vil References 1 Amijee H et al Inhibitors of protein aggregation and toxicity Biochemical Society Transactions 37 4 692 696 2009 Dedeoglu A et al Therapeutic effects of cystamine in a murine model of Huntington s disease The Journal of Neuroscience 22 20 8942 8950 2002 Wong E et al Autophagy mediated clearance of aggresomes is not a universal phenomeno
3. PB1 domain and can inter act with ubiquitinylated proteins via its C terminal domain Also p62 binds directly to LC3 and GABARAP family proteins via a specific sequence motif The protein is itself degraded by autophagy and serves to link ubiquiti nylated proteins to the autophagic machinery in order to enable their degrada tion in the lysosome Since p62 accumulates when autophagy is inhibited and decreased levels can be observed when autophagy is induced p62 may be used as a marker to study autophagic flux Figure 4 shows the use of a fluo rescein conjugated antibody directed to p62 in concert with the ProteoStat aggresome dye in co localizing these elements of the autophagic process in intact cells It is clear that p62 co localizes with aggresomes ProteoStat aggresome dye Fluorescein p62 antibody Compositeimage Figure 4 Aggresomes within HeLa cells previously treated for 12 hours with 5uM MG 132 detected by ProteoStat aggresome dye showing co localization with fluorescein p62 antibody as observed by fluorescence microscopy 10 2 Flow Cytometry Figure 5 shows the typical results of flow cytometry based analysis of cell populations using the ProteoStat aggresome red detection kit Uninduced control and 5 uM MG 132 treated Jurkat cells T Cell leukemia were used After 18 hours treatment cells were loaded with ProteoStat aggresome red detection reagent then analyzed without washing by flow cytometry
4. addition of the cell suspension Allow to incubate for 30 minutes on ice Collect the fixed cells by centrifugation at 800 x g for 10 15 minutes Pour out the supernatant being careful not to lose the cell pellet Wash the cells by re suspending the cell pellet with 2mL 1X PBS using a micropipet Then transfer the cell suspension through the cap of a BD tube with cell strainer cap BD 352235 to remove cell debris Then centrifuge the tube at 800 x g for 10 15 minutes Re move the strainer cap and carefully pour out the supernatant being careful not to lose the cell pellet Re suspend the cell pellet in 500 uL of freshly diluted 5 000 10 000 fold ProteoStat Aggresome Red Detection Reagent see step V A5b page 4 It is important to achieve a monodisperse cell sus pension at this step by gently pipetting up and down repeatedly Protect samples from light and incubate for 30 minutes at room temperature Analyze the samples in the FL3 channel of a flow cytometer No washing is required prior to the flow cytometry analysis CALCULATION OF RESULTS a Obtain the mean fluorescence intensity MFI values for treated and untreated sample b Calculate the aggresome propensity factor APF value using the following formula APF 100 x MF lireated MF lcontrot MF ltreated The expected APF value using the control MG 132 is greater than 25 VI APPENDICES A FLUORESCENCE CHANNEL SELECTION FOR DATA COLLECTION T
5. and permeabilized cells The detection reagent supplied in the ProteoStat Aggresome Detection Kit becomes brightly fluorescent upon binding to aggregated proteins within vesicles produced during aggresome formation and has been validated under a wide range of conditions in which the autophagy and proteasome pathways are known to be modulated MG 132 a proteasome inhibitor is included as a positive control in the kit A nuclear counterstain is provided in the kit as well to highlight this organelle This kit can be used to facilitate understanding of the basic molecu lar processes involved in the four key steps of autophagosome dependent degradation namely induction or cargo packaging vesicle formation and completion docking and fusion and vesicle breakdown The assay is poten tially applicable to the identification of small molecules that inhibit aggresome formation as well as immuno localization studies between aggregated protein cargo and the various proteins implicated in aggresome formation such as histone deacetylase 6 parkin ataxin 3 dynein motor complex and ubiquilin 1 Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored at lt 20 C protected from light When stored properly these reagents are stable for one year from date of receipt Avoid repeated freezing and thawing Reagents provided in the kit are sufficient for approximately 100 assays for flow cytometry applicatio
6. the cells Carefully re suspend and wash the cells with 2 mL of 1X PBS Remove the supernatant and carefully re suspend the cells in 2 mL 1X PBS Centrifuge for 5 minutes at room temperature at 400 x g Carefully remove most of the supernatant by aspiration Using a micropipet 11 12 13 14 15 16 17 18 gently re suspend the cell pellet in the remaining 1X PBS 200 pL Using a micropipet add drop wise the cell suspension into 2mL of 4 formaldehyde solution see section V A3 page 3 contained in a 15 mL conical tube Slowly vortex the fixative during addition of the cell suspension Allow to stand for 30 minutes at room temperature Collect the fixed cells by centrifugation at 800 x g for 10 15 minutes Pour out the supernatant Do not aspirate CAUTION Fixed cells do not stick to the walls of the tube as tightly as live cells Be careful not to lose the cell pellet Re suspend the cell pellet in the remaining small volume of super natant using a micropipet Add 2 mL of 1X PBS to the cells and cen trifuge at 800 x g for 10 15 minutes Pour out the supernatant being careful not to lose the cell pellet Re suspend the cell pellet in the remaining small volume of super natant using a micropipet Using a micropipet add drop wise the cell suspension into 2 mL of permeabilizing solution see section V A4 page 3 contained in a 15 mL conical tube Slowly vortex the perme abilizing solution during
7. ERNA TONAL UK amp IRELAND ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK T 0845601 1488 UK customers T 44 0 1392 825900 from overseas F 44 0 1392 825910 E info ukGenzolifesciences com www enzolifesciences com www enzolifesciences com FRANCE ENZO LIFE SCIENCES c o Covalab s a s 13 Avenue Albert Einstein FR 69100 Villeurbanne France T 33 472 440 655 F 33 437 484 239 E info fr enzolifesciences com www enzolifesciences com Alexis assay designs Stressgen
8. L MICROCOPY ADHERENT CELLS 1 Grow the cells directly on the glass slides to a 80 confluency 2 Treat the cells with the compound s of interest and negative control cells with vehicle only 3 Prepare positive control cells by incubating with the diluted Protea some Inhibitor 5 10 uM MG 132 see section V A1 page 3 for 18 hours under normal tissue culture conditions 4 Carefully wash the cells twice with 200 uL of 1X PBS per 1 cm sur face area foreach wash a typical 2 to 4 well slide would require 100 uL 5 Carefully remove excess 1X PBS and dispense 200 uL of 4 formal dehyde per 1 cm surface area Incubate for 30 minutes at room temperature 10 11 12 Carefully wash the cells twice with 200 uL of 1X PBS per 1 cm sur face area for each wash Carefully remove excess 1X PBS and dispense 120 uL of Permeabi lizing Solution see section V A4 page 3 per 1 cm surface area Place slides on ice and gently shake them for 30 minutes Carefully wash the cells twice with 200 uL of 1X PBS per 1 cm sur face area for each wash Carefully remove excess buffer and dispense 200 uL of Dual Detec tion Reagent see section V A5a page 4 per 1 surface area to cover the monolayer cells Protect samples from light and incubate for 30 minutes at room temperature Carefully wash the cells with 200 uL of 1X PBS per 1 cm surface area Remove excess buffer and place coverslip on microscope slide Analy
9. LS 11 Troubleshooting Guide 12 Introduction In mammalian cells aggregated proteins may be concentrated by microtubule dependent retrograde transport to perinuclear sites of aggregate deposition referred to as aggresomes Aggresomes are inclusion bodies that form when the ubiquitin proteasome machinery is overwhelmed with aggregation prone proteins Typically an aggresome forms in response to some cellular stress such as hyperthermia viral infection or exposure to reactive oxygen species Aggresomes appear to provide a cytoprotective function by sequestering the toxic aggregated proteins and may also facilitate their ultimate elimination from cells by autophagy Certain cellular inclusion bodies associated with human disease are thought to arise from an aggresomal response including Lewy bodies associated with neurons in Parkinson s disease Mallory bodies associated with liver cells in alcoholic liver disease and hyaline inclusion bodies associated with astrocytes in amyotrophic lateral sclerosis Non physiological protein mutations or genetically engineered cell lines have been developed for assessment of the effects of protein aggregation within cells Enzo Life Sciences ProteoStat Aggresome Detection Kit contains a novel 488 nm excitable red fluorescent molecular rotor dye to specifically detect denatured protein cargo within aggresomes and aggresome like inclusion bodies in fixed
10. M into your culture medium and grow cells for 6 to 18 hours The agent has been validated with human cervical carci noma cell line HeLa human T lymphocyte cell line Jurkat and hu man bone osteosarcoma epithelial cell line U2OS Unused stock solution of MG 132 may be stored at 20 C for several weeks 1X Assay Buffer Allow the 10X Assay Buffer to warm to room temperature Make sure that the reagent is free of any crystallization before dilution Prepare enough 1X Assay Buffer for the number of samples to be assayed by diluting each milliliter mL of the 10X Assay Buffer with 9 mL of deionized water 4 Formaldehyde Solution The following procedure is for preparation of 10 mL of 4 formalde hyde solution Dilute 0 4 gram paraformaldehyde to a final volume of 10 mL with 1X Assay Buffer Mix well Permeabilizing Solution The following procedure is for preparation of 10 mL of permeabilizing solution 0 5 Triton X 100 3 mM EDTA pH 8 Add 50 uL Triton X 100 and 60 uL of 0 5M EDTA pH 8 to 9 89 mL of 1X Assay Buffer Mix well ProteoStat Aggresome Detection Reagent For optimal staining the concentration of the ProteoStat Aggresome dye will vary depending upon the application Suggestions are provided to use as guidelines though some modifications may be required depending upon the particular cell type employed in the application a For fluorescence microscopy prepare a sufficient amount of Dual Detection Reagent fo
11. ease cell density Fixed cells don t stick to walls of the tube as live cells Follow the proce dure described in the manual Precipitate is observed in the 10X Assay Buffer Precipitate forms at low temperatures Allow solution to warm to room temperature or 37 C then vortex to dissolve all precipitate MG 132 treated cells appear dead or are no longer attached to the plate surface The of MG 132 may differ with different cell lines 12 Try lowering the dose of MG 132 c2 Enzo Life Sciences www enzolifesciences com Enabling Discovery in Life Science NORTH SOUTH AMERICA ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler Pike Plymouth Meeting PA 19462 1202 USA T 1 800 942 0430 610 941 0430 F 610 941 9252 E info usaGenzolifesciences com SWITZERLAND amp REST OF EUROPE ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 441 061 926 89 89 F 441 061 926 89 79 E info chGenzolifesciences com www enzolifesciences com GERMANY ENZO LIFE SCIENCES GMBH Marie Curie Strasse 8 DE 79539 L rrach Germany T 49 0 7621 5500 526 Toll Free 0800 664 9518 F 449 0 7621 5500 527 E info deGenzolifesciences com www enzolifesciences com BENELUX ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium T 32 03 466 04 20 F 432 03 466 0429 E info be enzolifesciences com www enzolifesciences com incorporating NT
12. ese products are offered under a limited warranty The products are guaranteed to meet appropriate specifications described in the package insert at the time of shipment Enzo Life Sciences sole obligation is to replace the product to the extent of the purchase price All claims must be made to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo CELLestial and ProteoStat are trademarks of Enzo Life Sciences Inc Several of Enzo s products and product applications are covered by US and foreign patents and patents pending Contents I Introduction eii 1 ll Reagents Provided and Storage 2 Additional Materials Required 2 IV Safety Warnings and Precautions 2 V Methods and 3 REAGENT PREPARATION 3 B CELL PREPARATIONS cesse eene nenne 4 C CELL ANALYSIS BY FLUORESCENCE CONFOCAL MICROSCOPY ADHERENT 4 D CELL ANALYSIS BY FLUORESCENCE CONFOCAL MICROSCOPY SUSPENSION 5 E CELL ANALYSIS BY FLOW 6 VI Appendices nnn 8 A FLUORESCENCE CHANNEL SELECTION FOR DATA COLLECTION sse 8 B EXPECTED RESULTS 8 VII 2 eeE
13. he selection of optimal filter sets for a fluorescence microscopy application requires matching the optical filter specifications to the spectral characteristics of the dyes employed in the analysis see Figure 1 Consult the microscope or filter set manufacturer for assis tance in selecting optimal filter sets for your microscope Predesigned filter sets for Texas Red should work well for this application For flow cytometry fluorescence channel FL 3 is recommended for analy sis of the ProteoStat aggresome red dye staining using a 488 nm laser source 500 200 250 300 350 400 450 500 550 600 Wavelength nm Wavelength nm Figure 1 Excitation and fluorescence emission spectra for ProteoStat Aggresome dye ex em 500 600 nm panel A and Hoechst 33342 ex em 350 461 nm panel B All spectra were determined in 1X Assay Buffer B EXPECTED RESULTS 1 Microscopy Selective degradation of intracellular targets such as misfolded pro teins and damaged organelles is an important homeostatic function that autophagy has acquired in addition to its more general role in restoring the nutrient balance during stress and starvation Although the exact mechanism underlying selection of autophagic substrates remains the subject of much study ubiquitinylation is now appreciated to be a signal for autophagic degradation of aggregated proteins In order to better understand the nature and contribution of the pathway modulators to t
14. he specific targeting of protein cargo for degradation the ProteoStat aggresome red fluorescent molecular rotor dye has been developed specifically devised to detect aggregated proteins within the aggresome and aggresome like structures This detection reagent detects protein cargo accumulation within these structures as observed by fluorescence microscopy see Figure 2 Figure 2 ProteoStat aggresome dye detects protein accumulation within aggre somes as observed by fluorescence microscopy HeLa cells were mock induced with 0 2 DMSO panel A or induced with 5 uM MG 132 panel B for 12 hours at 37 C After treatment cells were incubated with ProteoStat aggresome dye for 30 minutes The ability to detect aggresomes was demonstrated using various potent cell permeable and selective proteasome inhibitors Lacta cystin Epoxomicin and Bortezomib US FDA approved drug Velcade as shown in Figure 3 Control Epoxomicin 0 5 Lactacystin 4M Velcade 0 5uM Figure 3 Aggresomes detected by ProteoStat aggresome dye in HeLa cells after overnight incubation with various proteasome inhibitors as observed by fluores cence microscopy The p62 protein also called sequestosome 1 SQSTM 1 is an ubiquitin binding scaffold protein that co localises with ubiquitinylated protein aggregates in for example many neurodegenerative diseases and proteinopathies of the liver The protein is able to polymerise via an N terminal
15. isperse cell suspension at this step by gently pipetting up and down repeatedly Protect samples from light and incubate for 30 minutes at room temperature Resuspend the cells in 2 mL of 1X PBS centrifuge them at 800 x g for 10 15 minutes and remove the supernatant Resuspend the cells in 100 uL of 1X PBS and apply a 20 pL aliquot of the cell suspension sufficient for 2 x 10 cells onto a microscope slide Immediately overlay the cells with a cover slip Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard Texas Red filter set for imaging the cell aggresome signal and a DAPI filter set optional for imaging the nuclear signal CELL ANALYSIS FLOW CYTOMETRY BOTH SUSPENSION AND ADHERENT CELL LINES 1 Cells should be maintained via standard tissue culture practice in a humidified incubator at 37 C 5 CO Make sure that cells are healthy and in the log phase of growth before starting an experiment Treat cells with compound of interest and negative control cells with vehicle Prepare positive control cells by incubating with the diluted Protea some Inhibitor 5 10 uM MG 132 see section V A1 page 3 for 18 hours under normal tissue culture conditions At the end of the treatment trypsinize adherent cells or collect cells suspension cells Samples may contain 1 x 10 to 2 x 10 cells per mL Centrifuge at 400 x g for 5 minutes to pellet
16. materi als should be treated as potentially hazardous and handled as such They should be disposed of in accordance with established safety procedures To avoid photobleaching perform all manipulations in low light environ ments or protected from light by other means 2 V Methods and Procedures The procedures described in this manual assume that the user is familiar with the basic principles and practices of flow cytometry and is able to run samples according to the operator s manual pertaining to the instrument being used NOTE Allow all reagents to thaw at room temperature before starting with the procedures Upon thawing gently hand mix or vortex the reagents prior to use to ensure a homogenous solution Briefly centrifuge the vials at the time of first use as well as for all subsequent uses to gather the contents at the bottom of the tube A REAGENT PREPARATION 1 Positive Control MG 132 has been shown to be a regulator of protein homeostasis inducing both the unfolded protein response the heat shock response 5 Proteasome inhibition induces accumulation of heat shock protein mRNA activation of heat shock proteins and enhanced thermotolerance in various cell types Inhibiting protea somes accelerates the formation of perinuclear aggresomes within cells Reconstitute the lyophilized MG 132 120 nmol in 12 uL DMSO for a 10 mM stock solution To use it as a positive control dilute the MG 132 to 5 10 u
17. n Human Molecular Genetics 17 16 2570 2582 2008 Mu T W et al Proteostasis regulators and pharmacologic chaperones synergise to correct protein misfolding diseases Cell 134 769 781 2008 Murakawa Y et al Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells Cancer Res 67 18 8536 8543 2007 BjerkeyG et al p62 SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin induced cell death J Cell Biol 171 4 603 14 Nov 21 2005 11 Vill Troubleshooting Guide Problem Potential Cause Suggestion ProteoStat aggresome red dye staining fails to stain live cells The dye is only suitable for fixed and or permeabilized cells Use the dye only in fixed and or permeabilized cells Low ProteoStat aggre some red staining in all treatments including posi tive control A low concentration of the ProteoStat Aggresome Detection Reagent was used or the reagent was incubated with the cells for an insufficient length of time Either increase the re agent concentration or increase the incubation time Cell viability is low Cells should be in log growth phase Avoid shear stress do not vortex and avoid exces sive formation of bubbles ProteoStat aggresome red dye stained cells are too low to be readily quan tified Cell density is either too low or cells were lost during processing Incr
18. n or 200 assays for fluorescence microscopy Reagent Quantity ProteoStat Aggresome Detection Reagent 10 uL Hoechst 33342 Nuclear Stain 50 uL Proteasome Inhibitor MG 132 120 nmol 10X Assay Buffer 25mL Additional Materials Required Flow cytometer equipped with 488 nm laser source Standard fluorescence microscope Tubes appropriate for holding cells for the flow cytometer Calibrated adjustable precision pipetters preferably with disposable plastic tips Adjustable speed centrifuge with swinging buckets for suspension cultures Deionized water Anhydrous DMSO Total growth medium suitable for cell type Glass microscope slides Glass cover slips 18 x 18 mm Paraformaldehyde EDTA pH 8 Triton X 100 1X Phosphate buffered saline 1X PBS Safety Warnings and Precautions This product is for research use only and is not intended for diagnostic purposes The ProteoStat Aggresome Detection Reagent contains DMSO which is readily absorbed through the skin DMSO is harmful if ingested or absorbed through the skin and may cause irritation to the eyes Observe appropriate precautions when handling these reagents Reagents should be treated as possible mutagens and should be handled with care and disposed of properly Observe good laboratory practices Gloves lab coat and protective eyewear should always be worn Never pipet by mouth Do not eat drink or smoke in the laboratory areas All blood components and biological
19. nutes at room temperature Collect the fixed cells by centrifugation at 800 x g for 10 15 minutes Pour out the supernatant Do not aspirate CAUTION Fixed cells do not stick to the walls of the tube as tightly as live cells Be careful not to lose the cell pellet Re suspend the cell pellet in the remaining small volume of super natant using a micropipet Add 2 mL of 1X PBS to the cells mix well and centrifuge at 800 x g for 10 15 minutes Pour out the super 5 11 12 13 14 15 16 17 18 natant being careful not to lose the cell pellet Re suspend the cell pellet in the remaining small volume of super natant using a micropipet Using a micropipet add drop wise the cell suspension into 2 mL of permeabilizing solution see section V A4 page 4 contained in a 15 mL conical tube Slowly vortex the permeabilizing solution during addition of the cell suspension Allow to incubate for 30 minutes on ice Collect the fixed cells by centrifugation at 800 x g for 10 15 minutes Pour out the supernatant being careful not to lose the cell pellet Re suspend the cell pellet in the remaining small volume of super natant using a micropipette and wash again using 2 ml 1X PBS Then centrifuge at 800 x g for 10 15 minutes Pour out the super natant being careful not to lose the cell pellet Re suspend the cell pellet in 100 uL of Dual Detection Reagent from step V A5a page 4 It is important to achieve a monod
20. r the number of samples to be assayed as follows For every 2 mL of 1X Assay Buffer see section V A2 page 3 or cell culture medium add 1 uL of ProteoStat Aggre some Detection Reagent and 2 uL of Hoechst 33342 Nuclear Stain NOTE a The dyes may be combined into one staining solution or each may be used separately if desired b The Hoechst 33342 Nuclear Stain can be diluted further if its staining intensity is much stronger than that of the ProteoStaf aggresome red dye c When staining BFP or CFP expressing cells the Hoechst 33342 Nuclear Stain should be omitted due to its spectral overlap with these fluorescent proteins b For flow cytometry dilute the ProteoStat Aggresome Detection Reagent 5 000 10 000 fold with 1X Assay Buffer or buffer of choice B CELL PREPARATIONS Cells should be maintained via standard tissue culture practices Always make sure that cells are healthy and in the log phase of growth before using them for the experiment Positive control cells should be pretreated with the MG 132 a cell permeable proteasome inhibitor for 6 18 hours Response to MG 132 is time and concentration dependent and may also vary significantly depending upon cell type and cell line Negative control cells should be treated with a vehicle DMSO media or other solvent used to reconsti tute or dilute an inducer or inhibitor for an equal length of time under similar conditions C CELL ANALYSIS BY FLUORESCENCE CONFOCA
21. ze the stained cells by wide field fluorescence or confocal mi croscopy 60X magnification recommended Use a standard Texas Red filter set for imaging the cell aggresome signal and a DAPI filter set optional for imaging the nuclear signal CELL ANALYSIS BY FLUORESCENCE CONFOCAL MICROSCOPY SUSPENSION CELLS 1 10 Cells should be cultured to a density not to exceed 1 x 10 cells mL Make sure that cells are healthy and in the log phase of growth before starting an experiment Treat the cells with the compound of interest and the negative control cells with vehicle Prepare positive control cells by incubating with the diluted Protea some Inhibitor 5 10 uM MG 132 see section V A1 page 3 for 18 hours under normal tissue culture conditions At the end of the treatment collect cells to ensure cell count of 2 x 10 to 4 x 10 cells sample Centrifuge suspension for 5 minutes at room temperature at 400 x g Remove the supernatant and carefully re suspend the cells in 2 mL of 1X PBS Centrifuge for 5 minutes at room temperature at 400 x g Carefully remove most of the supernatant by aspiration Using a micropipet gently re suspend the cell pellet in the remaining 1X PBS 200 uL Using a micropipet add drop wise the cell suspension into 2mL of 496 formaldehyde solution see section V A3 page 3 contained in a 15 mL conical tube Slowly vortex the fixative during addition of the cell suspension Allow to stand for 30 mi
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