User Manual-ENZ-51008-100
Contents
1. 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 ug mL can be made 1X Assay Buffer 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 Red 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 h2. 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 cell samples prepare fresh DNA Staining Permeabilizing Solution as follows NOTE A concentration of 5 20 uM Nuclear ID Red dye is recom mended for permeabilized cells The procedure described below is for preparation of 20 uM dye solution For each sample to be stained dilute 1 ul Nuclear ID Red Cell 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 5 x10 cells mL Incubate for 30 min at 37 C in the dark Analyze the samples in the PerCP Cy5 5 or FL3 channel of a flow cytometer with a 488 nm excitation laser For 633 nm excitation use appropriate collection filters gt 700nm FL4 or FL5 APC Cy7 channel COMBINING GFP ANALYSIS OR IHC WITH DNA STAINING The live cell Nuclear ID Red dye allows the simultaneous analysis of a cell population for other parameters as well such as fluorescent protein expression levels BFP CFP GFP and or YFP or immuno histochemistry IHC with coumarin or fluorescein labeled cell surface antibodies Analysis of intracellular targets by IHC requires permeabilization and fixation VI APPENDICES A Fluorescen
3. of cells in a given sample that are in Go G1 S and G M phases as well as to quantify cells in the sub G phase and 2 DNA studies in live permeabilized and fixed cells for normal cell lines 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 tw
4. Enzo GFP Certified Nuclear ID Red Cell Cycle Analysis Kit for Flow Cytometry Instruction Manual Cat No 51008 100 100 assays For research use only Rev 1 0 1 April 2009 Notice to Purchaser The GFP Certified Nuclear ID Red 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 microscopy flow cytometry and HCS where consistency and reproduci bility 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
5. Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo GFP Certified 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 TREO GU CUI ON occitano ll Reagents Provided and Storage rrrsrrnussvnnnnnvnnnnenn lll Additional Materials Required rnrnannvnnnnnnnnvnnnnnnnenn IV Safety Warnings and PrecautionS sr rassrnnnvvrnnnenn V Methods and Procedures nnnnnnnunnnnnannnnnnnnnnnnnnnnnnununen A REAGENT PREPARATION errrenvvvernrennvversrrnnnversrrnnnvnenr B CELL PREPARATIONS u220rsnsuesennennnnnnnnnnnnnnnnnnnn STAINING LIVE CELLS 00 terete STAINING ETHANOL FIXED CELLS ee STAINING PERMEABILIZED CELLS ee COMBINING GFP ANALYSIS OR IHC WITH DNA STAINING enter ene nmoo VI Appendices runnnsnnvnnnnnnnvnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnenr A FLUORESCENCE CHANNEL SELECTION FOR DATA COLLECTION eee eee eee tees B RESULTS sanset aa ei VII References nn VIII Troubleshooting Guide mmsrrnnnnvnnnnvvnnnnvnnnnnnnnnnvnnnnenn Introduction Enzo Life Sciences GFP Certified Nuclear ID Red Cell Cycle Analy sis 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
6. X 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 concentration of 5 20 uM Nuclear ID Red dye is recom mended for ethanol fixed cells The procedure described below is for preparation of 20 uM dye solution For each sample to be stained dilute 1 ul Nuclear ID Red 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 5 x10 cells mL Incubate for 30 min at 37 C in the dark 11 Analyze the samples in the PerCP Cy5 5 or FL3 channel of a flow cytometer with a 488 nm excitation laser For 633 nm excitation use appropriate collection filters gt 700nm FL4 or FL5 APC Cy7 channel 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 g 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 5 x 10 cells
7. ce Channel Selection for Data Collection Fluorescence channels suitable for Propidium iodide PerCPCy5 5 gt 650nm or FL3 channel are recommended for imaging Nuclear ID Red Detection Reagent with 488 nm excitation or for 633nm excitation use appropriate collection filters gt 700nm APC Cy7 FL4 or FL5 channels B 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 Red 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 Red dye intercalates exclusively into the base pairs of double 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 G 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 re
8. ch 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 other 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 to exceed 0 2 To prepare a 0 1 ug mL solution
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10. elve 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 Nuclear ID Red 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 Red 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 su
11. ining Solution from step 6 Incubate for 30 minutes at 37 C in the dark It is important to achieve a monodisperse cell suspension at this step by gently pipetting up and down repeatedly 8 Analyze the samples in the PerCP Cy5 5 PI or FL3 channel of a flow cytometer with a 488 nm excitation laser For a 633 nm excitation use appropriate collection filters gt 700 nm FL4 or FL5 APC Cy7 channel D STAINING ETHANOL FIXED CELLS 1 10 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 g 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 Red 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 1
12. is 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 demonstrates the ability of the Nuclear ID Red dye to reflect the DNA content and cell cycle distribution 1 G M Dean Jett Fox 1 RMS 16 59 G 10 55 m4 S 27 3 q G 62 17 2 G p 188 4 Gan 376 Figure 2 Effect of a cell cycle gm G CV 13 61 A z Gs CV 13 21 perturbation agent on DNA lt G1 1 38 synthesis in Jurkat cells The gt Gp 2 49 cells were treated with 0 1 ug mL Nocodazole for 20 hours Cells were then washed and stained with Nuclear ID Red Cell Cycle 20 un an 800 1000 FL3 Detection Reagent 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 measurement 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 generate
13. ours 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 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 5 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 20 40 uM Nuclear ID Red dye is recom mended for live cell DNA analysis The procedure described below is for preparation of 40 uM dye solution For each sample to be stained dilute 2 uL Nuclear ID Red 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 Sta
14. presenting G2 M phase cells which contain four copies of each chromosome and thus twice the dye content of Go G1 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 G G and G M peaks Dean Jett Fox RMS 27 62 G 51 35 S 30 99 G 17 16 a G p 193 3 Figure 1 Typical DNA profile 8 nn from asynchronously growing EN eve keg Jurkat cells Normally about 40 lt G 1 39 of the cells are in G phase and ER about 20 each in S and G M phases Using standard 1 D flow cytometry it is not possible to r separate G phase cells from W a 600 800 1000 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 CV gt _ 100 2 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 debr
15. s 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 1x 10 to 5 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 incubation 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
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