Expert Cytometry

Пікірлер

@snehasurya1034 19 күн бұрын

Hello sir I'm quite intrested to learn about flow cytometry and ur videos have helped me to understand more thank you

@Waga_Waga00 26 күн бұрын

Hi many thanks for the tips. I am struggling with the same problem as well. I'd like to ask if it is silanized or siliconized tube that was recommended?

@hrushikeshhrushi6492 3 ай бұрын

Thanks for this video, it made me to understand the concept of flow cell and hydrodynamic focusing clear.

@Antoine_Fainthearted 6 ай бұрын

Great quick explanation, helped me out a lot - thanks!

@JSEIjsei 6 ай бұрын

great information, thanks

@NguyenThaiHoangTam 9 ай бұрын

Should I add 1g NaN3 into 1L double distilled water for Sheath Fluid or no need? thank you for your advice!

@tommerson5121 10 ай бұрын

I know autof is used in retinal examinations. My question is whether the light used to excite the cells (laser) is safe for the retina. It would almost seem that those levels of hyperintense light might be phototoxic. Any thoughts? Thanks.

@job506 10 ай бұрын

Many thanks for this video. In my cell cycle analysis, the statistics that I obtained for the phases summed up being above 100%. Please, what can I do?

@rute9alger187 Жыл бұрын

Hello. Please can you help me in the use the emergency button of bd facs aria iii and thank in advance

@moreyfede Жыл бұрын

I have the same question as Jakub. A set of samples in which I use compbeads, and also the live dead dye. How to reconcile both compensations?

@kubaksiazkiewicz Жыл бұрын

What if I have a ump channel that includes a series of antibodies as well as live/dead stain?

@osamabeenlacking2987 2 жыл бұрын

On foe nem

@akritino7437 2 жыл бұрын

So you mean to say that when we use FMO + Isotype together, we may/may not see its off target binding. I have had a staining wherein FMO+ Isotype showed the same staining pattern compared to the sample with all the colours stained while the isotype alone gave no specific positive signal. So, is it wrong to do so in practice?

@krecikowi 2 жыл бұрын

Your videos are pure gold. It is like you know what you are talking about ;)

@Yanisdu 2 жыл бұрын

You saved me in a test. I'm so thankful. Greetings from Chile.

@nurlatifahmohdnor8939 2 жыл бұрын

T = E P = S M = F Hablur = Crystal

@shaaaja 3 жыл бұрын

Thanks a lot for this video it was explained very nicely and I could follow through! Currently doing my Bachelor Thesis and something just didn't sit right with me doing the subjective visual gating so I started looking for the right way to do it and found your video. Had a hard time understanding my software before but now it is much clearer! I will include these findings definitely in my method part! Greetings from Switzerland :)

@yasminebader5656 3 жыл бұрын

What do you mean with the linear dynamic range of the PM?

@julianikonova2424 3 жыл бұрын

Thank you! Now I start work with human FoxP3 and i have a big priblem with losing cells after permibialization. Can you help me with protocol washing cells?

@rahullovesthepayne8690 5 ай бұрын

Hello, I was just wondering if you have found the answer to your question lol

@SimonCU 3 жыл бұрын

I can understand why u used FMO for CD25 (external marker) but I don't understand why you did not use Isotype for the intracellular staining because for intracellular staining some FoxP3 antibodies might bind unspecifically to internal proteins. Usually intracellular staining I use an ISO flourochrome and the surface staining I use FMO. If you use isotype for your intracellular staining and FMO for your surface staining your gating will look a lot better. You can try it. That is why your FoxP3 FMO plot is so low... You need an isotype so that your cells move more positive a bit.

@JMSouchak 3 жыл бұрын

Below is the title of the paper mentioned and the PMID. Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages PMID: 27731950

@akrititrehan5136 3 жыл бұрын

Can you let me know about any resources for gating? I am new to gating and I'm not sure how to gate on lymphocytes population in one plot and get to different sub types of t cells in other plots. Please let me know how to gate in any dot plot figure. Thank you.

@nadaj.9943 3 жыл бұрын

Thank u, sir. This was really helpful!

@OmicsAcademy 3 жыл бұрын

Hi sir, could you please explain what gate is in mass cytometry? Thanks in advance.

@yanaheyvaert6964 3 жыл бұрын

How is the compensation matrix made? How does the P2 gate information contributes to it? I read that the P2 gate reveals the level of spillover of the fluorophore into other detectors, but we only measure the fluorescence of each fluorophore in one detector, eg FITC in FITC detector? I don't get what information the P2 gate brings..

@medicallab.scienceeducatio9452 4 жыл бұрын

thanks, That was informative

@shimaabelal5776 4 жыл бұрын

Is it possible to follow course to learn how to count bacteria using flow cytometer? When and how much?

@pekkuenliew6668 4 жыл бұрын

If I increase the number of cells to stain, do I have to increase the antibodies amount that I put in to stain? I think likely yes. Is there a certain formula to it?

@216emily 4 жыл бұрын

Thanks!

@stevenservin627 4 жыл бұрын

Thanks a lot!

@claudyaallicia1422 4 жыл бұрын

may I know why you did not set the gate for the negative peak for Violet G 550_40-A Live_Dead Yellow?

@kristineravelo4505 4 жыл бұрын

Thank you for the very informative videos! Do you happen to have one detailing the effects of angles on scatter or one that reviews statistical analysis for flow? For example, how to interpret RAS?

@howieseay8311 4 жыл бұрын

Nice work, Tim!

@camilanavarrete1106 4 жыл бұрын

Hi. I am from Chile and recently I started using cytometry in my experiments. I usually use two FITC and PE fluorophors. It has been difficult to compensate because I don't understand very well how it is done. You could record a step-by-step video of an experiment with two fluorophores to see the compensation. I would be very grateful.

@expertcytometry6235 4 жыл бұрын

Thank you for your feedback. The compensation process is the same for 2 colors as it is for 20 colors. Hopefully, the videos here will help.

@Tracks777 4 жыл бұрын

nice video

@kenwong237 4 жыл бұрын

When looking for the average for cells that are positive for phagocytosis do I look at the median or mean values?

@expertcytometry6235 4 жыл бұрын

That will depend on your question and the technology you are using. Generally, however, for expression/fluorescence the median is a better measure.

@kenwong237 4 жыл бұрын

@@expertcytometry6235 thanks

@mariahjordan4694 4 жыл бұрын

Why would a vector control be needed in image cytometry?

@expertcytometry6235 4 жыл бұрын

Mariah, can you please elaborate on what you mean by a vector control?

@anaifuentes8904 4 жыл бұрын

Thanks. How can I do Rmax in Moflo?

@expertcytometry6235 4 жыл бұрын

www.ncbi.nlm.nih.gov/pmc/articles/PMC4503806/ - The authors used a MoFlo for their studies, so I would check this out for the details of how they collected the samples.

@traptown7916 4 жыл бұрын

Expert Cytometry

@aditihariharan1866 4 жыл бұрын

Hi, Tim. Thanks for making this video! I just had a question related to cell flow: the pulse width of a cell can be approximated to its time of flight through the laser interrogation point. My question is this: Why is it that we get a larger pulse width for larger cells, and vice versa?

@expertcytometry6235 4 жыл бұрын

Hi, Aditi -- thanks for your question! I created a follow-up video that I hope answers it. kzfaq.info/get/bejne/naeXf7OW1ce3gZs.html Let me know what other questions you have!

@aditihariharan1866 4 жыл бұрын

@@expertcytometry6235 Hi, Tim. Thanks a lot! I actually still have a bit of a doubt: wouldn't the passage of individual cells through the laser interrogation point and the concurrent pulse characteristics obtained be determined by the flow speed of the fluidics, and not just the width of the interrogating laser beam?

@aditihariharan1866 4 жыл бұрын

And thus, wouldn't larger cells pass through the laser interrogation point faster than smaller cells for the same flow speed of the fluidics, giving a shorter pulse width for larger cells?

@expertcytometry6235 4 жыл бұрын

@@aditihariharan1866 Imagine a setup where an observer is on one side of a street and a light is on the other side. As an auto blocks the light, the observer starts a stopwatch and stops it when he can see the light again. The time on the stopwatch is a measure of the time the auto spends passing by the light. Now, if we have traffic at a constant speed, and a Smart car passes by, the observer measures a time of 3 seconds. The next vehicle to pass by is an 18-wheeler, which the observer measures a time of 10 seconds. This makes sense since the 18-wheeler is much larger than the Smart car. The same is happening in the flow cytometer. We have an observer (PMT/PD), and a light. Rather than a stop watch, we are observing the time from the start of the pulse to the end of the pulse. The larger the cell, the longer that transition will take. Since the flow rate is constant, the difference that is being observed is size dependent. Does that help clear things up?

@aditihariharan1866 4 жыл бұрын

@@expertcytometry6235 It does, quite a bit! But what I'd like to know exactly is whether or not the speed of fluid flow within the flow cytometer can be taken to be the speed of the individual cells passing through the laser interrogation point? Because the above example that you've used to explain the variation in pulse width with cell size would hold only if all the cells had the same speed when passing through the laser interrogation point, no?

@ommostafa1 4 жыл бұрын

thanks

@Redsg1995 4 жыл бұрын

Thank you so much. Excellent explanation!

@wenhuili4522 5 жыл бұрын

What can be done if there is long tail in the Q1 when gating

@Biomeducated 5 жыл бұрын

Excellent tips! You provide tons of value for researchers using Flow/FACS, thank you team ExCyte!

@expertcytometry6235 5 жыл бұрын

Thank you for your feedback.

@Biomeducated 5 жыл бұрын

Good introduction! Would you say that bead compensation is sufficient? Or do you always include single stain cell controls as well? I've heard this can be helpful for autofluorescent cells (maybe like monocytes/macrophages), but would you do this for certain cell lines as well?

@expertcytometry6235 5 жыл бұрын

In general, bead based compensation is sufficient. As part of the panel development phase, it is important to determine/confirm that the bead signal will meet the 3 rules of compensation. This is also the time to determine that the signal of the antigen is higher than the AF of cells. You may want to read: expertcytometry.com/set-monitor-optimal-voltages-flow-cytometry-experiment/

@Biomeducated 5 жыл бұрын

@@expertcytometry6235 Thanks so much for these insights!

@skyys1000 5 жыл бұрын

I recently got into flow cytometry job and I am loving your videos. Please keep up the good work!

@expertcytometry6235 5 жыл бұрын

Thank you for the feedback Yeon Kim,

@deliasotomunoz2122 5 жыл бұрын

Hi, thanks a lot for the super useful video! Would you mind sharing a link to the paper you mentioned? THANKS!

@expertcytometry6235 5 жыл бұрын

Delia Soto Munoz, you can find a lot of that information on this blog: expertcytometry.com/strengths-and-weaknesses-of-isotype-controls-in-flow-cytometry/

@antonjets 5 жыл бұрын

Hi Tim, would you mind touching up on the comment of "reducing the sensitivity of the channel". I understand resolution (SI) quite well, but could you give a definition for sensitivity. I see a lot of flow explanations combine "resolution sensitivity" together. I know this is a bit off topic to autoflourescence but I am glad you mentioned it.

@expertcytometry6235 5 жыл бұрын

Anthony, sensitivity is defined as the ability to separate the negative signal from the positive signal. We talk about resolution sensitivity in the context of can a given detector resolve the negative and positive populations, especially in the context of a polychromatic panel.

@antonjets 5 жыл бұрын

@@expertcytometry6235 Thanks Tim, so is it safe to say that resolution and sensitivity are the same thing. I attended a training course saying that resolution is "the ability to separate the negative signal from the positive signal"

@expertcytometry6235 5 жыл бұрын

@@antonjets in this context, yes.

@geneno1984 5 жыл бұрын

Did you have training videos for fc500

@expertcytometry6235 5 жыл бұрын

At the present time we do not have videos for that instrument

@geneno1984 5 жыл бұрын

Did you work on fc500

@expertcytometry6235 5 жыл бұрын

I have not worked on the FC500, but have colleagues who do. You can email us at [email protected]

Ең жақсы KZfaq

Пікірлер