Cell Cycle Analysis - Science method

I have given the MCF-7 treated cells and control for cell cycle analysis by FACs. Unstained control MCf-7 cell have produce a peak in cell cycle analysis. So this result is wrong? if it is wrong, where it might have gone wrong?

I performed a cell cycle analysis using the cell cycle platform in FlowJo and found that some samples showed a negative value in the Sub-G0 population (see attached image). While I understand this result doesn’t make sense biologically, I’m wondering how I should interpret it and what steps I could take to correct these values. Thanks

Hello everyone,

I'm studying cell cycle regulation in diseased MSC,I did BrdU cell cycle analysis,and there was significant delay in G1/S phase in diseased cells in comparison to normal cells

I want to analyze gene and protein expression of cell cycle regulatory factors

My question is how to make sure that all the cells at G1/S phase transition ?

Hi, I culture HACAT mammalian immortalized cells. I am interested to check the protein changes by western blot and cell cycle analysis by flow cytometry. Can i store the harvested cell pellets in -80 freezer without any storing reagent before performing western or flow cytometry?

Thank you

I am a newbie in FACS analysis: so I used this reagent from dojindo called cell cycle blue reagent and do staining in live cells (not like PI) they stated that fixation is not needeed, the problem is this reagent has not been used in available paper for my cell lines (min6 insulinoma cell lines), thus I don't know why my result is like this... I tried to analyse in FlowJo but I couldn't get the result of the cell cycle that is usually shown. 1st peak G0/G1, then s phase, then G2/M phase... what went wrong?

I attached some of my result:

I would like to do a FACS analysis with BrdU-7aad staining, how long should I incubate with BrdU if my cells doubling time is 48hours?

Hello everyone.

I have performed cell cycle analysis by FCM using FxCycle PI/RNase staining solution from Invitrogen for MSCs. I fixed my cells with 70% ethanol for 2 hours at 4 degree C before staining with the solution.

My results showed 2 peaks which commonly known as G0/G1 peak and also G2/M peak, however the G2/M peak was very far from the G0/G1 peak and not showing 4N DNA content (attached file).

What would be the probable causes?

I am thankful for any tips and advice.

I am searching for the best approach to statistically evaluate differences in cell cycle distribution between different cell populations. We are analysing the cell cycle distribution, by staining DNA with PI and analysing what percentage of the cell population is in the different phases (GO/G1, S and G”/M) using flow cytometry and the Flow Jo software. So we get percentages of cells in these three phases for different cell populations and we want to analyse differences between the cell cycle distributions for these populations (usually treated cells in comparison to control). Using simple ANOVA or ttest statistics is probably not the best here, as the amount of cells in each of the phases is co-dependent on the amount of cells in the other phases. What would be the best approach?

When I perform cell cycle analysis using Cell Quest flow cytometer, I can not well initial gating of FSC-H and SSC-H. Because most of the cells are not see in the FSC and SSC figures. If anyone have experience of initial gating in cell cycle using PI, please help me.

I need a positive control for a cell cycle analysis experiment. Can anyone please suggest how you would arrest some cells in G0 to be used as a control? Thank you!

Hi there, I would really appreciate some help. I have done the flow cytometry using this kit: BrdU Flow Kits http://www.icms.qmul.ac.uk/flowcytometry/uses/cellcycleanalysis/diagrams/brdu.pdf

As recommended, I use FITC anti-BrdU and 7-AAD for DNA staining and cell cycle analysis. Somehow my result looks like this and I am not sure what the problem is here since this is the first time I have done it.

I'm trying to do BrdU stainning in AML cell lines. first cells were incubated with brdu for 2 hr at 37c then after washing cells incubated with cold 70% ethanol in ice for 30 min followed by 2N HCl treatment for 30 min at RT. Before cells being incubated with FITC conjugated anti-Brdu, cells incubated with 2ml 0.1M borax (NaB4O7) pH 8.5, incubate for 5 minutes at room temperature. This protocol was used in our lab and gave a nice result. However, I did not got the good separation of cells in S phase from cells in G1 and M phase.

Does anybody had this problem before and have good suggestions or know why S phase cells did not go far from cells in other phases

 

Hello everyone,

I'm new to flow cytometer analysis.

For the first time, I did analysis for PI stained hela cells. but I got the histogram like this. I want to do cell cycle analysis. Can anyone help me to improve this?

Thank you

Dear Researchers, which is the RNase A solvent for cell cycle analysis? DEPC? PBS? or DMSO?

Hello Researchers, I have been trying to use DAPI for cell cycle flow experiment, however the result looked really funky, does anyone have any advice?

The mice that I used is WT B6/J mouse around 6 weeks old. I use MBI pan T cells kit to isolate T cells and stimulate them with CD3 (1:50) plate bound (2h) & CD28 (1:250) in solution and cultured cells for 24h. For media I used RPMI 1640 with 10%FBS + 1% L-glu and P/S.

Here is the protocol I have been using. Have around 5 million cells after 24h.

1. Label with 100uL of 1:200 ZOMBIE NIR diluted in PBS for 20 minutes at RT. Wash once with FACS buffer.

2. Label with 100uL of 1:250 APC diluted in FACS buffer for 20 minutes at 4C. Wash once with FACS buffer.

3. Fix cells by adding 1 mL of Foxp3 Fixation/Permeabilization working solution to each sample and vortex. Incubate for 30minutes at RT. Protect from light.

4. Centrifuge cells @1200rpm for 8 minutes at RT, resuspend cells in 1mL PBS, incubate 15minute at RT to rehydrate cells.

5. Centrifuge cells @1200rpm for 8 minutes at RT. Dilute DAPI to 3ug/mL in perm buffer, resuspend cells in 250uL DAPI perm buffer.

6. Incubate the samples for 15–30 minutes at room temperature, protected from light. Analyze the samples without washing.

I´m using a protocol in order to detect by flow cytometry (using a 488-nm laser and 530-nm/30 filter or similar) whether my cells are positive for B-galactosidase or not. In this protocol I have to fix the cells with paraformaldehyde to 2% in PBS during 10 minutes before incubating with the working solution. After detecting my cells positive and negative for B-galactosidase I have sorted these two populations of cells and I have performed a cell cycle analysis with propidium iodide (IP). As these cells were fixed with 2% paraformaldehyde, I have had to adapt the recommended protocol for the cell cycle analysis (fix with 70% ethanol and incubate with RNAase + IP) and instead of fixing with ethanol I have permeabilised my fixed cells with 0,25% triton x-100 during 15 minutes before incubating with IP. The problem is that when I have analysed the phases of the cell cycle, they are not distinguished as good as with the recommended protocol. I have also tried to look for the cell cycle with 7AAD but the results were even worst. Does anyone know how can I adapt my protocol in order to improve the cell cycle analysis in the cells already fixed with paraformaldehyde?

I am testing Prussian blue (Fe3+[Fe3+(CN)6]) cathode for Sodium ion battery.

Literatures showed 2 voltage plateau (while it is not that clear in voltage plateau, still contribute a bit in capacity) and higher capacity as shown first figures from

.

However, It seems like I only have one voltage plateau and not even half capacity as shown my data second figure. I used 2 - 4.5V for cycling. The material synthesis was same as the paper.

differences were

1. I used active material: super P: PVDF = 65:25:10. While literature used active material: acetylene black: PVDF = 80:10:10.

2. I used 0.3M KTFSI electrolyte. While literature used KPF6 in EC/DEC.

What could be the reason of having only one plateau?

Hi, I would like to obtain synchronised cultures of B. subtilis with an efficient and reproductible method. Has anyone done this before and have a clear idea of the best way to do it?

Thanks a lot four your help,

1. I'm gonna fix my cells for cell cycle analysis via flow cytometry method. I've just read a protocol from this link "https://www.abcam.com/protocols/flow-cytometric-analysis-of-cell-cycle-with-propidium-iodide-dna-staining". My question is how long can we keep cell in fixation using 70% ethanol solution?

2. I have 200 mg powder of cisplastin. Does anyone know how to prepare for stock and how to store the stock for long-term using?

Thank you so much!

My substance decreases percentage of cell numbers in the G1 and increases percentage of cell numbers in the G2 phase while percentage of cell numbers in the S phase(cell cycle) remains same compared to the saline control in the cytometry assay. WB shows unregulated expression of Cycline D1.What can this suggest? (Btw, CCK-8 assay show higher proliferation rate in treated cells)

I do not know which area should I gate in the area vs height graph to distinguish between the doublets and polyploid cells using flowcytometry? I am working with a Beckman Coulter Machine. I have done PI staining

I need to study the cell cycle using PI by FACS. I would like to know if the difference in the cell number between samples can affect the FACS analysis. In the scatter plot (Cell count vs PI) the histogram changes the position along the PI axis between the samples. Why? How can I have the histogram plot fixed along PI axis?

Hi everyone,

We are trying to optimize a protocole for cell cycle analysis in 6-well plates. We prefer this format of plates because it is what we use regularly for siRNA transfections. So we wouldn't like to change the size of plates.

We have a protocol using PI in which we fix the cells with ethanol. We lose almost 90% of cells after this fixation.

Could you give me tips for avoiding this loss of cells?

Thanks.

Dear all,

I am about to purchase a bench top flow cytometer and our budget allows us to buy either the Accuri C6 Plus (BD) or the NovoCyte (ACEA), both with 2 lasers and 4 colors.

I have been using BD flow cytometers for more than 15 years including FACS Calibur, Aria, Canto (BD), and the bench top from Partec and the Guava.

I appreciate your feedback regarding the Accuri C6 Plus versus the NovoCyte if you have used either or both of them, regarding easiness of operation, software, and any potential frequent problem that may occur.

My main applications are cell cycle analysis, apoptosis, protein expression, as well as some immunophenotyping at the moment.

Thank you

Eiman

I prepared my samples with ethanol, RNase and PI. However I could not use the Flow Cytometry machine because of an technical problem. I covered my samples with foil and put them in +4. As my cells are fixated I don't question them but I'm not sure about the stability of PI. I don't know if PI is stable for longer times.

I have been staining yeast cells for analysis of cell cycle using SYTOX green and getting good results.

The cells i'm now tiring to stain express GFP so SYTOX green wouldn't work as they emit at the same wave length.

I have tried using both SYTO 60 and DRAQ5 with little success.

SYTO60 seems to mainly stain mitochondria and DRAQ5 does not provide great separation between G1 & G2.

anyone knows about a better protocol for DRAQ5 (for now i've been diluting it 1:1000 in PBS and incubating for 15 min in room temp) or a better DNA stain emitting in far-red and works well with yeast?

I was performing wound healing assay seeding 300,000cells/well in 6 well plates with 2mL RPMI medium.

At 0hr after intervention, the cells looked like the first picture.

At 24hr after intervention, the cells looked like the second picture

at 42hr after intervention, the cells looked like the third picture.

The control (DMSO) group on the other hand, demonstrated morphologies of the fourth, fifth, and sixth pictures at 0hr, 24hr, and 42hr after intervention respectively.

The thing is, the duration of this entire transformation shortened to about 20~24hrs when I redid the experiment under the same conditions, but with 1mL medium (same amount of cells seeded, same drug concentration, same type of 6 well plate, just with half the medium)

We can clearly see morphological differences at 42hr (the third vs the sixth picture) between the intervention and control groups, and I'm curious as to what may be the cause of it.

BTW, cell cycle analysis via flow cytometry was also performed, and there doesn't seem to be much of a difference between the control and intervention groups, with no signs of increase in SubG1 upon intervention, which kind of dismisses apoptosis. I'm therefore suspecting the possibility of autophagy.

So does anyone know the morphological changes of PC3 cells during the process of autophagy? Or does this indicate something else going on that I haven't considered?

THANX!!! :)

I have recently started to work with Flowing Software 2.5.1, Unfortunately I am stuck in the analysis of the data, because I can not modify the range the default axis values of the dot plot. Basically, I can go from logarithm to linear scale, but, for example, I cannot change the minimum and maximum value of each exis.

Thanks in advance for any advice

I'm using Abcam 112116 cell cycle assay kit - Green Fluorometric to detect cell cycle for HCT116 and Caco2 (colorectal cell lines). I've performed an optimization run using the recommended protocol (Nuclear green 1:200), and I even included several nuclear green concentrations (1:400, and 1:600).

Cell scattering shows two populations for HCT116 (probably cell duplicates) and minimal separate scattering with Caco2 (for 1:200 only as recommended by the kit). Nevertheless, when I select a single cell population I can't seem to see a normal cell cycle, always one prominent peak is showing.

What could cause the disappearance of cell cycle?

Note the following:

- Untreated unstained samples were used to detect auto-florescence

- Untreated stained cells were used to detect normal cell cycle

- I've used a cytotoxic treatment below IC50 for 24 hrs duration

- Treated stained cells are being investigated for cell cycle arrest compared to untreated ones

- Floating treated cells were collected in the samples

- Floated untreated cells were NOT collected in the samples

- no starvation was done

- Incubation of cells with nuclear green was 50 minutes

- cells are adherent, so trypsinization was done and following by complete media wash prior to incubation of with nuclear green in fresh complete media

- I've used Doxorubacin as a sample, the graph shows shifting but I can't quantify it without distinct cell cycle phases (attached is a full profile scatter for dox).

Should any of the following suggestions be crucial for better cell cycle presentation:

- Starving the cells prior to treatment?

- Using short duration treatment (<24 hrs)?

- Using low passage cell line? avoid advanced passage cell lines?

- Using IC50 or above IC50 concentrations of cytotoxic agents?

- Using higher dye (nuclear green) concentration (>1:200; recommended is 1:200)?

- Using prolonged incubation time with the dye (>60 minutes; recommended is 30-60 minutes)?

- Using cell strainer to avoid duplicate cells?

- Floating cells in the treated/untreated samples should/not be collected?

Attached is a full scattering profile for the untreated HCT116 cell line. Note that the second peak is probably representing cell duplicates rather than a population of single cells in the G2 phase, despite using a cell strainer.

Thank you

any one know cyclin E inhibitor (Synthetic) . CDK2 inhibiotrs are there but i need cyclin E specific inhibitor.

Hello everyone, I would like to study how the silencing of a protein can affect the cell cycle using Control Cell Vs CRISPR-Cell KO for this protein. I would like to check the cell cycle using FACS and the PI staining . Could you suggest me a nice protocol describing some parameters to use ?

Thank's in advance.

Best regards !

Hi there,

I have been doing cell cycle analysis with PI/Flow Cyt for a few months now, and every so often, I get weird results (see attached pic) where I lost my G2 peak entirely, and my S-phase bleeds off from my G1 peak. I can never figure out what is wrong, but if anyone can give me some insight I would greatly appreciate it.

I harvest cells from a 6 well plate (70% confluency) - use accutase, not trypsin

I wash 2x with RT PBS, then I fix the cells using 400uL cold PBS and 800uL cold ETOH (everything added on ice)

Within the next few days, on the day I want to run Flow, I wash the cells again 1x PBS, put on 500uL of PI at 50ug/mL, incubate @37C for 30 mins, then put the cells in the fridge for up to 3 hours (or whenever the technician runs the Flow)

Sometimes I get great results, where I can see G1/S/G2, other times all my samples come out looking like the attached picture. Has anyone experienced this, and do you know why it happens?

I want to ask about the RNAse treatment for the cell cycle. What should be the ideal conditions during RNAse treatment i.e. Incubation period, temperature, time or either it should be placed in 5% CO2 incubator or not?

I would be using PI staining and flow cytometry to examine cell cycle arrest effects of a certain plant extract in MCF 7 cells. I now need a positive control causing cell cycle arrest at the G1/S phase. Do you have any ideas as to what kind of agent I should use for this purpose?

What will happen to cell cycle histogram in PI staining flow cytometry if RNase doesn't work? Does it change or move the histogram peaks?

Hi,

I currently have a problem which I cannot understand. I am fixing cells in 70% ethanol: cells are first trypsinized, spinned down and 70% ethanol in water is added dropwise while vortexing. Cells then stayed at -20C for about a week. Then, I spinned the cells at 500g for 10 min, and I saw clearly big nice pellets of fixed cells. Then, I removed the soup carefully, checked that the pellet is still there and then added cold PBS 1X, up-and-down and then spinned again at 500g, 10 min. But the pellet is not there! I removed the soup and kept it on the side and checked some of it under the microscope, no cells. I put new PBS to the first tube (that has the invisible "pellet"), up-and-down and again checked under the microscope, no cells there too! Could it be that my cells are bursting or something?

I want these cells for propidium iodide staining for cell cycle analysis in flow cytometry.

Does anyone who actually does this protocol have an idea? Could you provide specific speed and time for the spin steps?

Thanks

Hi, I am studying a protein that seems to be localized at cilia but only in very short times, so i want to see if it is in a specific cell cycle phase.

What markers could I use in immunofluorescence to identify the cell cycle phase?

I work with IMCD3 cell line, in which the ciliation is not synchronized (I am trying to work this out).

Thanks in advance for any suggestion.

Best,

Cecília

Hi,

I have been doing flow cytometry for cell cycle analysis using PI for a liver cancer cell line and I have been getting a big and strange peak where S-phase should be. Not sure if it's S-phase peak or a heterogenous population (where the peak represents the G1 phase). We had this cell lines STR tested and it was a 100% match and we have been using a low passage number (max P-17).

I couldn't find any literature having similar results. Any ideas what's happening here or how to get rid of it?

Thank you.

I have measured cell cycle progression of my cells by staining my cells with propidium iodine (PI). I obtained the histogram unlike what is expected with a high abundance of S phase in control cells and more cells in G2/M phase that expected.

It is the first flow cytometry experiment I did so I am wondering if I did something wrong to receive these data (protocol attached). Do you think it might have something to do with the cells handling?

I am using HEK293 cells but our Flow Cytometer (FC500 from Beckman Coulter) is mostly set up for the yeasts. We do not have any program set for the human cells and only one colleague of mine adjusted the program for HeLa cells.

I would really appreciate your feedback. Thank you in advance.

About to run cell cycle/apoptosis analysis on flow, i am unsure of which drug to use for my endothelial cell line. What i have now is etoposide and 5fu. and i am unsure of the concentration that is suitable for 72hrs treatment window. i have checked on pubmed and ncbi for clues but to no success. please help. thank you.

I have been running FACS analysis with a given protocol that does not use Triton X-100 treatment following fixation/permeabilization with 70% ethanol. The resultant cell cycle histogram has a huge G1 peak, but a poor S-phase and G2/M profile. Would we expect a difference using Triton X-100 in this protocol? Most protocols seem to use it, but some don't. My lab professor has never used it before and is reluctant to add it to the protocol.

Cancer treatment with three different concentrations has been used on cancer cell line for 24hr, and then ab139418 – Propidium Iodide Flow Cytometry Kit was used for cell cycle analysis. The attached file shows the results, where the DNA content in the second concentration (T2) is higher in G2 than S and G0.

Hello everyone

does any body know how can we analyse and gating the cell cycle data in G1,S and G2 phases in the cancer cell line by Flowjo software?

Hello, I want to perform FACS analysis to assess the cell cycle status in specific bone marrow cell populations by looking at the Ki67 against DAPI signal. We have working antibodies and DAPI solutions and I am troubleshooting the protocol.

In our last experiment, in one of our samples we observed a granular cell population that is DAPI high (comparable to signal in G2/S/M) but Ki67 negative/low (comparable to signal in G0). I know that all cells between G2/S/M phases according to DAPI signal should also be Ki67 positive/high.

My question then is, has someone experienced something similar while testing the protocol? I am wondering if it is a technical issue (eg buffer composition, DAPI concentration, machine voltages) or if this population can indeed come up in some cases. I wasn't able to find much information by quickly browsing the literature.

Thanks everyone!

We have a platform that can stimulate cells mechanically, and normally proliferation of cells decreases greatly with that stimulation.

So I thought that cells were arrested in G0 phase, but cell cycle analysis revealed decreased G0/G1 phase and increased S and G2/M phase.

Any possible explanations for this?

Hello,

What I want : I want to sort primary cells according to different cell cycle phases and then stain for transcription factors to image/flowcytometry/qPCR any of these.

Combining cell cycle analysis as well as trascription factors for flow would be the best.

has any one tried ?

Thanks.

Do I have to treat RNase A when measuring cell cycle by FACS? I am measuring cell cycle of Arabidopsis mutants by FACS. Because of a little expensive price of RNAse A, if it is not necessary, I would like to skip RNase treatment. Do you think I don't need to treat RNase?

I was trying to do cell cycle analysis using MCF-7 cell lines in BD-Flow cytromery instrument. The peak of all phages are shifting towards right of treated cells as compared to normal MCF cells. How to analyse this observation. Please share your ideas, if anybody has expertise on this.

Thanks

I have discordant findings using DAPI and propidium iodide. Should I trust any over the other? I study human memory CD4 T cells.

I have observed that when I pulse cells with EdU for analysing cell cycle kinetics, the pattern of staining is as in Fig.1. Considering the heterogeneity of S phase cells during the pulse, cells that are in early S phase appear as EdU[medium] DNA[2n] (marked by the blue box); cells that progress through S phase appear as EdU[high] DNA[2n-4n] (green box). However, cells that are in the late S phase must appear as EdU[med] DNA[4n], which I do not see (red box). I have pulsed cells for different durations (2h, 24h, 48h) and still observe similar distributions. I use propidium iodide for measuring DNA content.

Fig.2 is from the manufacturer's website. BrdU images from publications also show similar trends.

I am working on cell cycle analysis in keratinocytes and SCCs. My treatment is supposed to lock the cells in G2 phase. We are using flow cytometry to analyze the cells, with BrdU vs Propidium iodide as stains.

The first few times I did the experiment, it worked just fine, you could see distinct groups of G1, S, and G2 in the flow data.

However, this last time I ran the experiment, most of the samples (control and treated) had a big blob stretching foughly across where G1 and G2 should have been. My gut says that the cells probably were growing, but for whatever reason either the BrdU wasn't incorporated, or that the antibody did not bind effectively to it.

The only other thing I can think of is that the cells were dead during the BrdU treatment phase, but then they looked normal and attached under the microscope prior to harvesting them.

The only variation in my protocol this last time, was a slightly lengthened step where the samples sat at room temperature incubating with the anti-BrdU antibody. Normally this would be done for only 2 hours, but here they were done at 2:30-3 hours. Otherwise, the experiment was done as normal. I would imagine this would have allowed for better binding, not less.

I'm just a little stumped. During the BrdU process, all the cells had all the same reagents (I mixed enough for all samples with each step) and did all things at the same time, and yet some ended up worse off than others.

Any ideas or advice?

Thanks!

I did cell cycle analysis of AGS cell line by using flow cytometry (BD) after treatment with paclitaxel. But I am not familiar with flow cytometry. So after getting the histograms, I could not differentitate G0/G1, S and G2 phases (attached figures). Please give me guidance how I could get differentiation of phases.

After staining the cells with TB do I need to wash them in order to obtain the clear solution of cell suspension or the colored solution can be injected as such into flow cytometer? Also is it important to have argon laser for such experiments (I think not, because the laser is same just the source that generates it is different)?

Hi,

Guys, I performed cell cycle analysis using PI staining recently. The problem is the compound I am working should lead to cell cycle arrest and the data suggests that. But, this is not enough. (Data is attached; D24 and D48 compound treated samples for 24 and 48 hours).

What I see is that in compound treated cells and non-treated cells, the majority of cells are G1 phase. I can indeed see increase in cells in G1-phase in compound treated cells (suggesting G1/S arrest). But, the cells in G1 phase originally is too high already (nearly 70%).

Can someone suggest me error in my protocol?

1. Seed cells (24 hours)

2. Serum starve cells (24 hours)

3. Add compound treatment and DMSO (negative control) 24 or 48 hours)

4. Wash cells with PBS 2X

5. Treat cells with Trypsin-EDTA and collect cells and centrifuge at 300g for 5 minutes.

6. Remove media and add 4-5 mL (PBS+2%FBS )

7. Fix cells by adding 3-4 mL 70% ethanol. Make sure to vortex mildly while adding ethanol. Carefully check if they are no clumps being formed. Make sure to use pipette for continuous aspiration. Incubate for 1-2 hours at 4C on ice. Cells could be kept at -20C for weeks.

8. Add more PBS+2%FBS and centrifuge at 500g for 5 mins.

9. Wash 1 more time in PBS+2%FBS and centrifuge for 5 mins at 500g.

10. Add solution with 0.5 ug/ml RNAase in PBS (with 0.1% sodium citrate and 0.1% Triton-X 100) for 30 mins at 37C. Total solution volume is 0.5 mL

11. Add PI staining solution (100 ug/mL) 0.5 mL solution and keep at 4C overnight. It is suggested to vortex gently in between a couple of times.

Regards,

Pratik

I'm trying to combine Analysis of Apoptosis and Cell Cycle Analysis using flow cytometry. I'm using mouse multipotent neural progenitor or stem-like cells (C17.2) and have access to a BD Canto II machine (3 lasers/ 8 colors). I'm very new to flow cytometry.

I've already tested the combination Annexin V and PI staining - Stain the live cells with Annexin V, fix in 70% ethanol and stain with PI. The Annexin V staining wasn't sufficient in our adherend cells and the needed fixation (we used 70% ethanol).

Do you have any suggestions on how to combine these Analysis (in terms of dyes or fixative...) or is it better to do two seperate staining?

The data sheet says that it intercalates DNA, but I'm not sure if it does in a stechiomoetric way...

Hi There,

I have attempted to perform a double-thymidine block on my cells using a 24h first inc.-9h release-16 second inc. and 16-9-16 incubation periods in FUCCI DLD-1 cells. I haven't seen any synchronization. Does anyone have any recommendations or tips?

I am not sure if fluorescence microscopy by nuclear staining with DAPI / Hoechst dyes alone could be helpful in detecting genomic instability characteristics by fluorescence microscopy. Of course, one can perform cell cycle/ploidy anaysis. Please let me know if there is another simple experiment to assess genomic instability induced by a drug candidate.

Hi,

How long I need to starve MDA-MB-231 cells in serum free media to synchronize them before I infect them with my protein of interest using Adenovirus..

I'm aiming at conducting cell cycle analysis as end assay

published papers time range from 2-24hr?!

Thank you

I am doing cell cycle analysis using Propidium iodide dye in flow cytometry. My samples are

1) cells

2)cells+ silica

3) cells+ drug

4) Cells+ silica-drug

The cells treated for 24 hrs with the drug and silica-drug to the cells. The dead cells start floating after 24 hrs treatment. My doubt is that if I throw the media from the flask, dead cells also gone with the media. Whether I need to take the floating cells (dead) for the cell cycle analysis? Please note: my cells are adherent cells.

Dear all,

I want to determine the fraction of dead (or apoptotic) eGFP expressing cells in comparison to the fraction of dead non eGFP expressing cells. I have a BD LSR II available but I am a total beginner in FACS. However, I think that should be possible to descriminate in FACS.

What dyes should I use to label dead or dying cells? eGFP in combination with propidium iodide? eGFP in combination with DAPI? Which filters are appropriate and what pitfalls should I consider?

Thanks in advance!

I am currently working on antiangiogenic activity of natural bioactive compound. From my MTT result, IC50 is determined at 2ug/ml. Apoptosis occurred at 20 ug/ml but not at the IC50 level. However, when doing cell cycle analysis, no significant difference was observed between negative and treatment group (0.2, 2 and 20 ug/ml).

1. Could I say that the compound is anti-proliferative?

2. Is my cell cycle analysis making any sense?

Many thanks in advance for your help.

Cell cycle analysis

I have done cell cycle analysis of sychronized HEK-293 cells incubated with some nanomaterials and in their absence (control) The results show almost no difference in G1 phase, while a decrease in S phase, accompanied by an increase in G2/M phase appears. Could somebody give me some more insight into this data?

* cell cycle analysis was performed three times and these are the mean values. Sample 1 is the lower tested concentration and sample 2 is the higher tested concentration.

increased s phase in teh cell cycle should also be complemented with an increased g2m phase?

in context to cell cycle analysis using PI ?

we performed clonogenic assay by just seeding the cells at very low density and observed lesser formed colonies of our transfected cells compared to mock. However, similar cell cycle analysis was observed in both cells. What does it mean? Also the number of medium and large colonies was decreased in transfected cells compared to mock cells

We synchronize cells for different experiments, but my question is in reference to drug treatment. If we synchronize cells by minimal or no media, should we add the treatment or drug in complete or serum containing media?

If not, how would cells enter in cell cycle then? and if we dont want them to enter into active cycle, are we looking at the effect of drug or any molecule in cells at G0/G1 arrest?

How would it justify chemotherapeutic effect?

Any thoughts are welcome. 

Hello!

My substance decreases the percentage of cell numbers in the G2/M phase (cell cycle) compared to the saline control in the cytometry assay. What can this suggest? 

I'm currently analyzing cell viability using an Annexin V flow cytometry assay. I'm trying to determine if there is a statistically significant difference in live cells between my untreated control cells and cells dosed with different concentrations of a chemical. 5,000 cells are counted each time, with five replications per concentration. Again, live cells are reported as a percentage, and I'm trying to determine if there is a statistically significant difference in the average of percentage of live cells after five replications in control vs. treatment groups.