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RynDggn's Content

There have been 14 items by RynDggn (Search limited from 03-April 19)


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#124443 Single stain control for 293 cell line

Posted by RynDggn on 28 November 2011 - 08:30 PM in Flow Cytometry

In order to do color compensation, you don't need to use the 293 cells, you could use antibody capture beads which have a species specific capture antibody on the bead surface that you can stain with the CD47 PE antibody. You can use this for purposes of compensation.



#124442 sample pool

Posted by RynDggn on 28 November 2011 - 08:28 PM in Flow Cytometry

This is not so much a flow cytometry question, but a biological question. if you were taking spleen cells from litter mates as a control and didn't have enough cells, then it would probably be fine to pool samples and stain together. However, I would question whether you really have enough cells or not. See a paper from Mario Roederer entitled, How many events is enough? Are you Positive? http://onlinelibrary...to.a.20549/full



#124441 data analysis for single cell size and distribution

Posted by RynDggn on 28 November 2011 - 08:24 PM in Flow Cytometry

What sort of instrument are you using? Since traditional flow cytometers use light scatter, and since light scatter is based more so on refractive index and not size, it is impossible to convert the FSC and SSC axes to absolute um values. All you can do is make relative judgements based on the scale values (this group of cells is 2 times bigger than the other). It should be completely sufficient to compare your samples using this relative scale.



#124440 Extracting DNA from FACS sorted cells

Posted by RynDggn on 28 November 2011 - 08:21 PM in Flow Cytometry

The solution to the problem was already put forth by "almost a doctor." split the collected volume and spin. The reason this happens has been a point of conjecture for years. The best explanation I've heard of goes as follows. All the droplets containing cells that are deposited in your collection tube share the same charge. This build-up of charge causes the cells to be held up in solution and not pellet. One way people have tried to get around this is to transfer the liquid to another tube and therefore dissipate the charge. You could also insert a copper wire and connect it to something metal to act as a ground and allow the charge to flow out of the suspension.



#124439 FACS Sorting and Protein Purification

Posted by RynDggn on 28 November 2011 - 08:14 PM in Flow Cytometry

It's not so much the change in nozzle size but the reduced system pressure that is used when you increase the nozzle size. Typically a 70um tip utilizes a system pressure of 50-70PSI, while a 100um tip uses a pressure of 20-30 PSI. This decrease in pressure leads to a decrease shear stress experienced by the cells and typically leads to increased viability. In addition to this, you need to make sure you are collecting your cells into a well buffered collection media (such as HBSS + 1% BSA + 50mM HEPES). Since the PBS in the system is under high pressure, it tends to get acidic. If your cells are sensitive to this decreased pH, then they will die. However, if you add HEPES to your collection buffer, you can neutralize the pH and increase viability.

Regarding the Trypsin thing. We use a product called Accutase, which is also pretty gentle on cells.



#124438 FlowJo on Mac - biexponential adjustment

Posted by RynDggn on 28 November 2011 - 08:08 PM in Flow Cytometry

My cells were acquired on BD FACSDiva, and I'm using a width basis of -100. Under Workspace Preferences I have ticked the box to "Allow Custom Visualisation".


Herein lies the problem. FACSDiVa uses a width basis default of -100, while FlowJo uses a default of -10. I do know you can change the width basis in the preferences using the button called 32-bit data (or something like that).



#124437 CFSE does not show distinct peaks

Posted by RynDggn on 28 November 2011 - 08:06 PM in Flow Cytometry

Cerise, your CFSE staining looks fine. Actually it looks pretty darn good. You'll never get separate peaks and analysis of such data requires modeling such as what is available in software like ModFit (verity software house) and FlowJo (Treestar).



#124436 staining at 37 degrees or RT - OK or really bad ?

Posted by RynDggn on 28 November 2011 - 08:02 PM in Flow Cytometry

The reason for keeping the cells on ice is to reduce internalization of the antibody. If cells are metabolically active and going through their normal surface antigen turnover, you could internalize antibodies which could lead to increases in background. One way to go about your staining procedure would be to stain the chemokine receptors 1st, wash, then do subsequent surface staining on ice or 4C and then wash again.



#124435 Help reading/identifying part of a FCM chart.

Posted by RynDggn on 28 November 2011 - 07:51 PM in Flow Cytometry

Well, according to the figure, they're labeling the first peak as 1C (i.e. 1 copy of DNA a.k.a. G1 cells) and the second peak as 2C (i.e. 2 copies of DNA a.k.a. G2/M cells). I'm sort of surprised this figure is published in Nature, as the staining looks really bad. Cell Cycle peaks are typically very sharp, whilst these peaks are very broad. This is usually the result of poor fixation or insufficient staining concentration/duration.



#124434 What if I froze a PE-conjugated antibody

Posted by RynDggn on 28 November 2011 - 07:47 PM in Flow Cytometry

Antibodies are sure to be fine when frozen and subsequently thawed. Many fluorochromes are also fine, including PE, most of the Alexa dyes, and FITC. Some that don't fair too well freezing are tandem dyes, such as PECy7 and APCCy7. Also, as with most things, you want to avoid repeated freeze/thaw cycles.



#124433 what's more useful in flow cytometry

Posted by RynDggn on 28 November 2011 - 07:45 PM in Flow Cytometry

If you're asking about laser lines, well then the 594nm laser is more useful. Not only can it be used to excite fluorochromes like Texas Red and Alexa 594, it can also excite APC and APC-tandems efficiently. So, you can get more bang for your buck with a 594nm. If you're talking about emission maxima, then 594nm and 589nm are a bit too close to differentiate, unless they were the emission maxima of two fluorochromes that were excited by two different lasers on a system with spatially separated beams. In this case, it would be possible to differentiate the two fluors.



#124432 Fixed cells, sticky cells?

Posted by RynDggn on 28 November 2011 - 07:42 PM in Flow Cytometry

Cells are not necessarily stickier upon fixation, but cells that are already clumping together will become permanently stuck together upon fixation. the PFA crosslinks proteins in close proximity. Whether these are proteins on a single cell's surface, or proteins in close proximity between two different cells is of no consequence.

Regarding the pelleting issues. It's likely you're fixing your cells in the presence of a buffer containing protein (such as BSA or FCS). Make sure to wash your cells with plain PBS before PFA fixation. Just like on the cells, the PFA will crosslink proteins in the buffer making the solution denser and therefore the cells don't pellet as well.



#124431 what's that small peak prior to G1?

Posted by RynDggn on 28 November 2011 - 07:36 PM in Flow Cytometry

The peak that is calculated as 52% is the G1 peak (at channel 200). G2/M is then around channel 400. The peak below the G1 is generally referred to as the Sub G1 peak and is indicative of cells that have undergone some DNA degradation due to apoptosis. One way to confirm this would be to display the DNA content on the x-axis and then forward scatter on the y-axis. The Sub G1 cells should be both low in the DNA content axis and low on the FSC channel. For more information on Sub G1, you can see: Kajstura M, Halicka HD, Pryjma J, Darzynkiewicz Z. Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete "sub-G1" peaks on DNA content histograms. Cytometry A 2007; 71A:125-131



#124429 What positive control for intracellular staining?

Posted by RynDggn on 28 November 2011 - 07:31 PM in Flow Cytometry

When people need to ensure their cells are properly permeabilized and in good condition, they'll sometimes use an antibody to F-Actin, which is present in all cells. If the F-Actin staining is always positive then at least you know that the cells are properly permeabilized and that your buffers are working properly. This could serve as a positive control.




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