User Manual-ENZ-51014-100
Contents
1. Nuclear ID Green Cell Cycle Detection Reagent Nocodazole Control 10X Assay Buffer Additional Materials Required Flow cytometer 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 optional Total growth medium suitable for cell type 70 Ethanol 10 Triton X 100 in water Safety Warnings and Precautions This product is for research use only and is not intended for diagnos tic purposes The Nuclear ID Green Cell Cycle 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 materials 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 environments or protected from light by othe2. procedure described below is for preparation of 10 uM dye solution For each sample to be stained dilute 1 uL Nuclear ID GreenCell Cycle Detection Reagent and 10 uL of 10 Triton X 100 to a final volume of 500 uL with media or buffer of choice Re suspend each fixed cell sample in 0 5 mL freshly prepared DNA Staining Permeabilizing Solution from step 6 to a final concentration of 1 x 10 to 1x10 cells mL Incubate for 30 min at room temperature 22 C or 37 C in the dark Analyze the samples in the FL1 GREEN channel of a flow cytometer with a 488 nm excitation laser VI APPENDICES A Fluorescence Channel Selection for Data Collection FL1 GREEN channel is recommended for imaging Nuclear ID Green Detection Reagent with 488 nm excitation Expected Results Propidium iodide staining of DNA in permeabilized cells and subse quent detection by flow cytometry has been widely employed to determine the percentage of cells in each phase of the cell cycle While prodium iodide is impermeable to plasma membrane and nuclear membranes Nuclear ID Green dye freely enters cells allowing cell cycle analysis without the need for laborious permeabili zation and fixation steps Propidium iodide additionally has the disadvantage that it stains all double stranded nucleic acids so cells must be incubated with RNase to remove any double stranded RNA Since Nuclear ID Green dye intercalates exclusively into the base pairs of double
3. Enzo Nuclear ID Green Cell Cycle Analysis Kit for Flow Cytometry Instruction Manual Cat No 51014 100 100 assays For research use only Rev 1 0 May 2009 Notice to Purchaser The Nuclear ID Green Cell Cycle Analysis Kit is a member of the CELLestial product line reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for live cell analysis applications CELLestial reagents and kits are optimal for use in demanding imaging applications such as confocal micros copy flow cytometry and HCS 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 These 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
4. V _ 100 Peak Channel In the above equation the peak channel is the mean channel number of the peak The smaller the CV of the peaks in the DNA histogram the more accurate is the measurement of ploidy and the better the estimation of the percentage of cells in the different parts of the cell cycle It is essential that any unnecessary broadening of the peaks due to misalignment of the instrument or suboptimal dye to DNA ratio should be minimized The number of cell clumps and the amount of debris present are also important factors influencing peak broadness The microtubule inhibitor Nocodazole can arrest the cell cycle at the G2 M phase Flow cytometric analysis Figure 2 below demon strates the ability of the Nuclear ID Green dye to reflect the DNA content and cell cycle distribution Dean Jett Fox RMS 10 86 300 G 17 62 S 18 88 Ga 55 73 G u 196 13 Gz u 366 39 Figure 2 Effect of a cell cycle SES perturbation agent on DNA Gz CV 8 7 d P ee synthesis in Jurkat cells The gt G2 0 12 cells were treated with 0 1 pg mL Nocodazole for 20 hours Cells were then washed and stained with o 200 400 600 800 1000 Nuclear ID Green Cell Cycle FITC A Detection Reagent N o o No Cells o o Vil References 1 Darzynkiewicz Robinson and Crissman eds 1994 Flow Cytometry Methods in Cell Biology 41 amp 42 Academic Press Inc San Diego 2 Nunez 2001 DNA m
5. days of receipt of order Trademarks and Patents Enzo and Nuclear ID 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 1 Introd ction geesde ee ll Reagents Provided and Storage uusnsnseenennnnnnnen lll Additional Materials Required ccssssseeseeee IV Safety Warnings and Precautions cceeeee V Methods and Procedures c0sssssscsssseesssssseessseeees A REAGENT PREPARATION see eeee eee eeeees B CELL PREPARATIONS cece ee eee eeeeenee ees C STAINING LIVE CELLS 00 eee D STAINING ETHANOL FIXED CELL E STAINING PERMEABILIZED CELLS ee VI Appendices nunssennseennnnnnennnnnnennnnannnnnnnnannennnnennnnn A FLUORESCENCE CHANNEL SELECTION FOR DATA COLLECTION B ESPECTEDRESULTS ceeeeeeeeseeeeeeeeee MIL References iii ic VII Troubleshooting Guide uuuuresusnnnnnnnnnnnnnnnnnnnnnnn Introduction Enzo Life Sciences Nuclear ID Green Cell Cycle Analysis Kit provides a convenient approach for studying the induction and inhibition of cell cycle progression by flow cytometry The kit is suitable for 1 determining the percentage of cells in a given sample that are in Go G S and G2 M phases as well as to quantify cells in the sub G phase prior to apoptosis and 2 DNA studies in live permeabilized and fixed cells for normal cell line
6. easurement and cell cycle analysis by flow cytometry Curr Issues Mol Biol 3 3 67 70 Vill Troubleshooting Guide Problem Potential Cause Suggestions DNA histograms are of poor overall quality The key elements in obtaining a histogram of high quality are good sample preparation correct cell to dye ratio and proper instrument alignment Live cell histo grams are more challeng ing to generate than fixed cell ones e Make sure the cell preparation protocol generates single cells with minimal clumping Filtration or trituration of cell suspensions may be required e Systematically vary the dye to cell ratio e Optimize for cell type medium or buffer used time of incubation temperature of incubation e Gate on the live cells and in crease the number of cycle events being analyzed e Verify that the flow cytometer is correctly aligned with stable fluidics using calibrated fluores cent beads of known CV e Verify that the cell concentration is 1 x 10 to 1 x1 0 cells mL e For permeabilized cells ensure that sufficient Triton X 100 has been added to the staining solu tion to permeabilize the cells e For ethanol fixed cells ensure that the cells were fixed and stored correctly No Gz block observed in cells treated with Nocodazole Incorrect drug concentra tion or time of incubation for cell type e Optimize for cell type medium or buffer used time of inc
7. oncentration of 5 20 uM Nuclear ID Green dye is recommended for ethanol fixed cells The procedure described below is for preparation of 10 uM dye solution For each sample to be stained dilute 1 uL Nuclear ID Green Cell Cycle Detection Reagent to a final volume of 500 uL with media or buffer of choice Re suspend each fixed cell sample in 0 5 mL freshly prepared DNA Staining Solution from step 9 to a final concentration of 1 x 10 to 1 x10 cells mL Incubate for 30 min at room temperature 22 C or 37 C in the dark Analyze the samples in the FL1 GREEN channel of a flow cytometer with a 488 nm excitation laser E STAINING PERMEABILIZED CELLS 1 Grow cells overnight to log phase in a humidified incubator at 37 C 5 CO Treat cells with or without experimental compounds Prepare positive control cells by incubating with pre diluted Nocodazole 0 1 0 2 ug mL see section A 1 page 3 for 18 24 hours under normal tissue culture conditions At the end of treatment trypsinize adherent cells or collect cells suspension cells Adjust cell count to 1 x 10 to 1 x 10 cells mL Centrifuge at 400 x gfor 5 min to pellet the cells Re suspend in media or other buffer of choice and centrifuge as before Immediately prior to staining the cell samples prepare fresh DNA Staining Permeabilizing Solution as follows NOTE A concentration of 5 20 uM Nuclear ID Green dye is recommended for permeabilized cells The
8. p and down repeatedly D 8 Analyze the samples in the FL1 GREEN channel of a flow cy tometer with a 488 nm excitation laser STAINING ETHANOL FIXED CELLS 1 10 11 Grow cells overnight to log phase in a humidified incubator at 37 C 5 CO Treat cells with or without experimental compounds Prepare positive control cells by incubating with pre diluted Nocodazole 0 1 0 2 ug mL see section A 1 page 3 for 18 24 hours under normal tissue culture conditions At the end of treatment trypsinize adherent cells or collect cells suspension cells Centrifuge at 400 x g for 5 min to pellet the cells washing twice with 1X Assay Buffer Resuspend the pellet in 100 200 uL 1X Assay Buffer This helps to prevent clumping Slowly add ice cold 70 Ethanol 20 C drop wise to each sample 10 cells mL while vortexing At this point cells may be stored at 20 C for several months before staining Cells must be in fixative for at least one hour prior to staining When ready to stain the cells with Nuclear ID Green Cell Cycle Detection Reagent centrifuge the fixed cells at 400 x g for 5 min Decant fixative thoroughly Wash cells by adding 5 mL of 1X Assay Buffer to each sample and gently resuspend the pellet Centrifuge the cells for 5 min at 500 x g and carefully remove the supernatant Immediately prior to staining the ethanol fixed cell samples prepare fresh DNA Staining Solution as follows NOTE A c
9. r means 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 Nocodazole a cell cycle perturbation agent is an anti neoplastic agent which exerts its effect by depolymerizing microtubules causing arrest in the G2 M phase of the cell cycle It may be used as a positive control for cell cycle distribution changes The Nocodazole provided in the kit is supplied at a stock concen tration of 1 mg mL in DMSO It is recommended that treatment with the agent be performed using 0 1 0 2 ug mL 1 2 uL stock per 10 mL medium in order to observe changes in nuclear morphology and that the final DMSO concentration in the assay not exceed 0 2 To prepare a 0 1 ug mL solution add 1 uL stock Nocodazole Control per 10 mL medium Use 2 uL stock Nocodazole Control per 10 mL medium for a 0 2 ug ml solution If small volumes are required an intermediate dilution e g 10
10. s and cell lines exhibiting multiple ploidy levels The progression of the cell cycle is controlled by a complex interplay among various cell cycle regulators that either stimulate or inhibit the cell from entering each stage of the cell cycle These regulators activate transcription factors which bind to DNA and turn on or off the production of proteins that result in cell division Dysfunction of any step in this regulatory cascade causes abnormal cell proliferation which underlies many human pathological conditions such as cancer A crucial step to understanding these conditions is the ability to understand the mechanisms underlying alterations in cell cycle progression A control cell cycle perturbation agent Nocodazole is provided for moni toring changes in cell cycle dynamics Potential applications for live cell studies are in the determination of cellular DNA content and cell cycle distribution for the detection of variations in growth patterns for monitoring apoptosis and for evaluating tumor cell behavior and suppressor gene mechanisms 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 at least twelve months Avoid repeated freezing and thawing Reagents provided in the kit are sufficient for approximately 100 assays using either live permeabilized or fixed cells Reagent Quantity
11. stranded DNA no RNase treatment is required and the fluorescence intensity of a stained cell is directly proportional to the DNA content in the cell Cells in the Gy G phase are either quiescent or preparing to enter the S phase DNA synthesis and contain one set of chromosomes An exponentially growing population of cells displays a DNA content distribution containing two major peaks a narrow peak representing Go G phase cells and a second smaller peak representing G2 M phase cells which contain four copies of each chromosome and thus twice the dye content of G G phase cells Figure 1 Cells in S phase are in the process of DNA replication and hence their DNA content distribution will fall between the Gol and G2 M peaks G0 G Dean Jett Fox RMS 24 03 G 44 2 S 34 5 Go 18 13 G p 191 42 1000 800 Gop 345 55 Figure 1 Typical DNA profile 3 A See from asynchronously growing 6 400 Bee al Jurkat cells Normally about gt G 0 69 40 of the cells are in G phase and about 34 in S and 20 in G M phases Using standard 1 D flow cytometry it is not 0 200 400 600 soo 1000 possible to separate G2 phase FITC A cells from mitotic cells The quality of a DNA histogram is estimated from the width of the peak of DNA from cells in G4 of the cycle This is measured by the coefficient of variation CV across the peak and is calculated from the standard deviation SD SD C
12. treated with a vehicle DMSO media or other solvent used to reconstitute or dilute an inducer or inhibitor for an equal length of time under similar conditions C STAINING LIVE CELLS 1 Grow cells overnight to log phase in a humidified incubator at 37 C 5 CO Treat cells with or without experimental compounds Prepare positive control cells by incubating with pre diluted Nocodazole 0 1 0 2 ug mL see section A 1 page 3 for 18 24 hours under normal tissue culture conditions At the end of treatment trypsinize adherent cells or collect cells suspension cells Adjust cell count to 1 x 10 to 1 x 10 cells mL Centrifuge at 400 x g for 5 min to pellet the cells Re suspend in media or other buffer of choice and centrifuge as before Immediately prior to staining the live cell samples prepare fresh DNA Staining Solution as follows NOTE A concentration of 5 20 uM Nuclear ID Green dye is recommended for live cell DNA analysis The procedure described below is for preparation of 10 uM dye solution For each sample to be stained dilute 1 uL Nuclear ID Green Cell Cycle Detection Reagent to a final volume of 500 uL with media or buffer of choice Resuspend each live cell sample in 0 5 mL of freshly prepared DNA Staining Solution from step 6 Incubate for 30 minutes at room temperature 22 C or 37 C in the dark It is important to achieve a monodisperse cell suspension at this step by gently pipetting u
13. ubation temperature of incubation NOTES www enzolifesciences com Enzo Life Sciences North South America ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler Pike Plymouth Meeting PA 19462 1202 USA Tel 1 800 942 0430 610 941 0430 Fax 610 941 9252 info usa enzolifesciences com Switzerland amp Rest of Europe ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland Tel 41 061 926 89 89 Fax 41 061 926 8979 info ch enzolifesciences com Benelux ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium Tel 32 0 3 466 04 20 Fax 32 0 3 466 04 29 info be enzolifesciences com Germany LIFE GmbH Marie Curie Strasse 8 DE 79539 Lorrach Germany Tel 49 0 7621 5500 526 Fax 49 0 7621 5500 527 into de enzolifesciences com incorporating UK amp Ireland ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK Tel 0845 601 1488 UK customers Tel 44 0 1392 825900 overseas Fax 44 0 1392 825910 info uk enzolifesciences com For Local Distributors please visit our Website BIOCHEMICALS LEXIS BIOMOL
14. ug mL can be made 1X Assay Butter NOTE The 1X Wash Buffer will be used for staining of fixed cells See section D page 5 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 DNA Staining Solutions The concentration of Nuclear ID Green dye for optimal staining will vary depending upon the application Suggestions are pro vided to use as guidelines though some modifications may be required depending upon the particular cell type employed and other factors such as the permeability of the dye to the cells or tissues To reduce potential artifacts from overloading the cells the concentration of the dye should be kept as low as possible Refer to sections C D and E for details on the preparation of the staining solution for specific applications Prepare sufficient amount of the staining solution for the number of samples to be assayed B CELL PREPARATIONS Cells should be maintained via standard tissue culture practices Positive control cells should be pretreated with Nocodazole Control for 18 24 hours Response to the perturbation agent Nocodazole is time and concentration dependent and may also vary significantly depending upon cell type and cell line Negative control cells should be
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