Flow Cytometry Discussion Troubleshooting forum on flow cytometry http://www.protocol-online.org/forums Thu, 09 Feb 2012 01:03:13 +0000 3600 Propidium iodide staining http://www.protocol-online.org/forums/topic/24346-propidium-iodide-staining/
Could anyone please tell me what concentration of PI I should use to stain dead cells before running FACS? It seems to vary from protocol to protocol... My stock concentration is 1mg/ml.

Thanks!]]> Thu, 09 Feb 2012 01:03:13 +0000 http://www.protocol-online.org/forums/topic/24346-propidium-iodide-staining/ Analysis of high throughput screen for cell surface markers http://www.protocol-online.org/forums/topic/24234-analysis-of-high-throughput-screen-for-cell-surface-markers/
I'm totally new to the world of flow cytometry data analysis and am looking for some help. I have 4 cell lines that were analyzed by high throughput screen flow cytometry for over 300 CD and other surface molecules. I received an excel file with MFI and %gate+ for each molecule for each cell line. I want to compared the level of expression between the cell lines. My questions are:

1) How do I normalized the data (I do not have a control or anything)
2) Do I use MFI or %gate+ for the anaylsis. For either, what is a good cut-off value (i.e. what is background level?)
3) How do I normalize the data?

Any insight would be greatly appreciated!]]> Tue, 31 Jan 2012 20:12:41 +0000 http://www.protocol-online.org/forums/topic/24234-analysis-of-high-throughput-screen-for-cell-surface-markers/ FACS positive control for Tuj1/beta3-tubulin http://www.protocol-online.org/forums/topic/24213-facs-positive-control-for-tuj1beta3-tubulin/
does anyone of you know a possible FACS positive control for an anti-human Tuj1 (=beta3-tubulin) antibody, apart from human CNS tissue which is difficult to obtain?

Thanks !]]> Mon, 30 Jan 2012 18:41:59 +0000 http://www.protocol-online.org/forums/topic/24213-facs-positive-control-for-tuj1beta3-tubulin/ IgG control http://www.protocol-online.org/forums/topic/24182-igg-control/
e.g.
sample 1: anti-human mouse monoclonal (IgG2) CD49f-APC
neg control: anti-human mouse monoclonal IgG-APC

sample 2: anti-human mouse monoclonal (IgG1) CD34-biotin (with SA-PE secondary Ab)
neg. control: anti-human mouse monoclonal IgG-PE

sample 3: anti-human mouse monoclonal (IgG2a) CD24 (with anti-mouse secondary Ab-FITC)
neg. control: anti-human mouse monoclonal IgG-FITC

Correct?

Also, how do you choose a company when you buy your IgG control? It probably won't matter too much, but I am just curious.

Thanks!]]> Fri, 27 Jan 2012 22:40:23 +0000 http://www.protocol-online.org/forums/topic/24182-igg-control/ Annexin binding buffer (urgent advice) http://www.protocol-online.org/forums/topic/24079-annexin-binding-buffer-urgent-advice/ I am new in flow cytometry and now one in my lab has any experience in it too,
I want to prepare annexin binding buffer, I found its concentration, but what I cant find are these concentration per not.
Could any one verify this meaning for me please.
I have attached the protocol of my Annexin-V fluos the reagent I have and it is written that I should have a binding buffer :
10mMole HEPEs.
140mMole NaCl, and 5mMole CaCl2.
my question these quantities per liter or what????
Sorry for silly question, but it made me confused.
Thank you in advance
Shimaa]]> Thu, 19 Jan 2012 05:10:06 +0000 http://www.protocol-online.org/forums/topic/24079-annexin-binding-buffer-urgent-advice/ receptor number and rate of internalization http://www.protocol-online.org/forums/topic/24004-receptor-number-and-rate-of-internalization/ how do you measure the number of receptors on a cell surface and receptor internalization rate, i.e., the turnover rate of receptors on a cell surface by flow cytometry? Thu, 12 Jan 2012 16:10:05 +0000 http://www.protocol-online.org/forums/topic/24004-receptor-number-and-rate-of-internalization/ ROS detection using DCFDA http://www.protocol-online.org/forums/topic/23965-ros-detection-using-dcfda/ Although application of NAC, a well known antioxidant significantly counteract the effect of my chemical, i always suffer one problem that the chemical shows no obvious influence on ROS level when using DCFDA or DHE to detect. It is certain that DCFDA works since the basic level of ROS was detectable. Is it possible that my chemical induces extraordinary ROS than the normal one?

The detail of experiment procedure is as follows:

Cells cultured in RPMI supplemented with 10% FBS and 1% P/S were treated with my chemical for 24 hours. Cells were washed with PBS, trpsinized, and neutralized with FBS. After washing with PBS again, cells were incubated in PBS containing 10 uM DCFDA for 30 min. After washing again, cells were analyzed using flow cytometry.

I had also tried to stain cells without trypsinization; however, the result remain similar with no significant difference.

Any suggestion?

Thank You]]> Tue, 10 Jan 2012 11:02:52 +0000 http://www.protocol-online.org/forums/topic/23965-ros-detection-using-dcfda/ FACS antibody titration http://www.protocol-online.org/forums/topic/23963-facs-antibody-titration/
I will be titrating my FACS antibodies (and this is my first time). Can anyone please answer some very basic questions below?

- Which cells should (or can) I use to titrate? (I will be sorting primary cells from normal tissue and/or normal epithelial cell line. Can I use ANY cells?)
- Is there any case where Fc-blocker is not required?
- Does the procedure somewhat vary depending on conjugation status (flourochrome, biotin, or non-conjugated), and if so, why?
- Do you recommend serial dilution or adding different amount of antibody to the same volume of buffer?

Thanks!]]> Tue, 10 Jan 2012 06:35:21 +0000 http://www.protocol-online.org/forums/topic/23963-facs-antibody-titration/ Dylight 650 help http://www.protocol-online.org/forums/topic/23938-dylight-650-help/
We are going to start using dylight 650 for some flow cytometry, but I can't find any quenching solutions that would work for this far wavelength (Ex/Em: 652/672)and I'm not sure if I could use trypan blue. anyone?
Happy new year!!!]]> Fri, 06 Jan 2012 23:34:12 +0000 http://www.protocol-online.org/forums/topic/23938-dylight-650-help/ Cytokine assay with cell supernatants HELP!!!! http://www.protocol-online.org/forums/topic/23928-cytokine-assay-with-cell-supernatants-help/
I am new (both Forum and cytokines!!).

We are trying to detect cytokines from cultured/stimulated PBMC.
We are incubating in triplicate for 5 days.

Do you pool your supernatants or Not?

Currently, we are not pooling the supernatant and running each individual well with a CBA and we are observing quite variations between wells.
We only run them once.

So what are your best practices?

Pooled supernatants and run in duplicates?
Unpooled supernatants and run once?


Thank you very much for your help!!!!]]> Fri, 06 Jan 2012 16:23:59 +0000 http://www.protocol-online.org/forums/topic/23928-cytokine-assay-with-cell-supernatants-help/ GFP tagged protein expression detection http://www.protocol-online.org/forums/topic/23788-gfp-tagged-protein-expression-detection/ differentiate cell containing one GFP linked protein expressing plasmid from two GFP linked protein expressing plasmid just by FACS.
Thanx.]]> Fri, 16 Dec 2011 07:44:22 +0000 http://www.protocol-online.org/forums/topic/23788-gfp-tagged-protein-expression-detection/ centrifugation speed/cell prep for flow http://www.protocol-online.org/forums/topic/23750-centrifugation-speedcell-prep-for-flow/
I am just wondering if there is a particular significance when prepping cells for flowcytometry to use relatively low centrifugation speed to spin cells down (usually at 300g)? The reason i ask is because I find that I am losing cells over the many times I need to wash/rinse cells centrifuge and aspirate supernatant. I fixed my cells in 1% paraformaldehyde overnight at -20C first and then processing for TUNEL assay. I wonder then if the problem because I did not fix it properly or was it because the centrifugation speed was just not high enough? If anyone has any insight, I'd really appreciate your input!]]> Tue, 13 Dec 2011 04:52:37 +0000 http://www.protocol-online.org/forums/topic/23750-centrifugation-speedcell-prep-for-flow/ ANNEXIN V staining for FLOW http://www.protocol-online.org/forums/topic/23708-annexin-v-staining-for-flow/
any input is appreciated...thanks in advance for your help!]]> Thu, 08 Dec 2011 20:49:43 +0000 http://www.protocol-online.org/forums/topic/23708-annexin-v-staining-for-flow/ Tween-20 vs Triton-X vs Saponin http://www.protocol-online.org/forums/topic/23604-tween-20-vs-triton-x-vs-saponin/
I know Triton is stronger/harsher vs Tween.

And would you have a separate permeabilisation step with detergent and then remove detergent from the antibody incubation buffer & wash buffer?

Apparently it keeps the membrane pores open? or anyone know the mechanism of action of detergents in immuno?]]> Tue, 29 Nov 2011 21:27:17 +0000 http://www.protocol-online.org/forums/topic/23604-tween-20-vs-triton-x-vs-saponin/ Serum in flow buffer http://www.protocol-online.org/forums/topic/23603-serum-in-flow-buffer/
And should we have detergent in the buffers as well?

Do you incubate your antibodies with a different buffer from what you use to wash them?]]> Tue, 29 Nov 2011 21:18:21 +0000 http://www.protocol-online.org/forums/topic/23603-serum-in-flow-buffer/ What positive control for intracellular staining? http://www.protocol-online.org/forums/topic/23585-what-positive-control-for-intracellular-staining/ I need to learn well technique of intracellular staining of PBMC on flow cytometry in order to make sure that later, when I do not see protein of my interest, the reason is that this protein is not expressed by the cell not because I am not able to make the procedure the correct way. But, I do not know what primary antibody, against what protein to buy and use. What intracellular protein I shall use to train detection by FCM to be 100% positive it is always expressed in PBMC...?

Thank you!
Paja]]> Mon, 28 Nov 2011 19:21:44 +0000 http://www.protocol-online.org/forums/topic/23585-what-positive-control-for-intracellular-staining/ <![CDATA[what's that small peak prior to G1?]]> http://www.protocol-online.org/forums/topic/23554-whats-that-small-peak-prior-to-g1/
I am just wondering if anyone of you have ever seen such a cell cycle pattern like this before for a mammalian cell line. This is untreated cells so I only expect to see a G1 and G2/M peak but I got an extra peak that I've never seen before in another batch of the same cell line or different cell line. Any opinion is hugely appreciated.

Attached File(s)

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]]> Thu, 24 Nov 2011 23:53:16 +0000 http://www.protocol-online.org/forums/topic/23554-whats-that-small-peak-prior-to-g1/ Fixed cells, sticky cells? http://www.protocol-online.org/forums/topic/23546-fixed-cells-sticky-cells/
I also find that my PFA-fixed cells do not pellet down as well after centrifugation for the washing steps?]]> Thu, 24 Nov 2011 09:24:33 +0000 http://www.protocol-online.org/forums/topic/23546-fixed-cells-sticky-cells/ Difference between flow cytometry and immunostaining? http://www.protocol-online.org/forums/topic/23497-difference-between-flow-cytometry-and-immunostaining/
Would trypsinising cells for the purposes of immuno-positivity staining in flow disrupt cellular structure integrity?]]> Tue, 22 Nov 2011 08:31:11 +0000 http://www.protocol-online.org/forums/topic/23497-difference-between-flow-cytometry-and-immunostaining/ <![CDATA[what's more useful in flow cytometry]]> http://www.protocol-online.org/forums/topic/23480-whats-more-useful-in-flow-cytometry/ which wavelength is more useful in flow cytometry? 594nm or 589nm? Mon, 21 Nov 2011 06:20:10 +0000 http://www.protocol-online.org/forums/topic/23480-whats-more-useful-in-flow-cytometry/