Hi !
I'm new in FACS analysis and I have some basics questions. I want to determine transfection efficiency of a cell population transfected with a fluorescent transgene (dsRed). I fixed the cells with PFA before FACS analysis.
For FACS detection I learned that a gate should be applied to detect "normal events", I mean to select a certain amount of non apoptotic, single viable cells. (e.g 10 000 events in this gate) Then I applied another gate on this population to discrimintae positive from negative fluorescent cells. This paret is OK, but the pb is the detection. Should I select all the cells to be analyzed or Should I restrict the population to the viable and single ones?
Thanks for help
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#2
gfischer
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Posted 25 March 2009 - 08:35 AM
BiotechAngel, on Mar 24 2009, 05:00 AM, said:
Hi !
I'm new in FACS analysis and I have some basics questions. I want to determine transfection efficiency of a cell population transfected with a fluorescent transgene (dsRed). I fixed the cells with PFA before FACS analysis.
For FACS detection I learned that a gate should be applied to detect "normal events", I mean to select a certain amount of non apoptotic, single viable cells. (e.g 10 000 events in this gate) Then I applied another gate on this population to discrimintae positive from negative fluorescent cells. This paret is OK, but the pb is the detection. Should I select all the cells to be analyzed or Should I restrict the population to the viable and single ones?
Thanks for help
I'm new in FACS analysis and I have some basics questions. I want to determine transfection efficiency of a cell population transfected with a fluorescent transgene (dsRed). I fixed the cells with PFA before FACS analysis.
For FACS detection I learned that a gate should be applied to detect "normal events", I mean to select a certain amount of non apoptotic, single viable cells. (e.g 10 000 events in this gate) Then I applied another gate on this population to discrimintae positive from negative fluorescent cells. This paret is OK, but the pb is the detection. Should I select all the cells to be analyzed or Should I restrict the population to the viable and single ones?
Thanks for help
First off, I don't think you really need to fix the cells before FACS, in fact it may even cause a problem. Regarding your query, I'd limit your data to viable cells, though others may disagree.
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#3
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Posted 26 March 2009 - 05:36 PM
gfischer, on Mar 26 2009, 12:35 AM, said:
BiotechAngel, on Mar 24 2009, 05:00 AM, said:
Hi !
I'm new in FACS analysis and I have some basics questions. I want to determine transfection efficiency of a cell population transfected with a fluorescent transgene (dsRed). I fixed the cells with PFA before FACS analysis.
For FACS detection I learned that a gate should be applied to detect "normal events", I mean to select a certain amount of non apoptotic, single viable cells. (e.g 10 000 events in this gate) Then I applied another gate on this population to discrimintae positive from negative fluorescent cells. This paret is OK, but the pb is the detection. Should I select all the cells to be analyzed or Should I restrict the population to the viable and single ones?
Thanks for help
I'm new in FACS analysis and I have some basics questions. I want to determine transfection efficiency of a cell population transfected with a fluorescent transgene (dsRed). I fixed the cells with PFA before FACS analysis.
For FACS detection I learned that a gate should be applied to detect "normal events", I mean to select a certain amount of non apoptotic, single viable cells. (e.g 10 000 events in this gate) Then I applied another gate on this population to discrimintae positive from negative fluorescent cells. This paret is OK, but the pb is the detection. Should I select all the cells to be analyzed or Should I restrict the population to the viable and single ones?
Thanks for help
First off, I don't think you really need to fix the cells before FACS, in fact it may even cause a problem. Regarding your query, I'd limit your data to viable cells, though others may disagree.
PFA would quench your some fluoresence molecules, at least FITC or GFP based on my experience. And you should always analyzed the viable and single cells in FACS, unless you care about the cell apoptosis in your story. May it help you and best wishes.
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#4
BiotechAngel
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Posted 27 March 2009 - 01:35 AM
First off, I don't think you really need to fix the cells before FACS, in fact it may even cause a problem. Regarding your query, I'd limit your data to viable cells, though others may disagree.
[/quote]
PFA would quench your some fluoresence molecules, at least FITC or GFP based on my experience. And you should always analyzed the viable and single cells in FACS, unless you care about the cell apoptosis in your story. May it help you and best wishes.
[/quote]
Thanks for the answers. I am currently working with dsRed instead of GFP . Does anyone have experience about PFA fixation on dsRed expressing cells??
Wow, thanks for your explanation but the point is why single cells only?? My boss thinks that aggregrates have to be analyzed too. Because if cells in aggregates are efficiently transfected and knowing that aggregates of our cells can easily be destroyed by pipetting, these cells might be very precious for next steps.
... other opitnion???
[/quote]
PFA would quench your some fluoresence molecules, at least FITC or GFP based on my experience. And you should always analyzed the viable and single cells in FACS, unless you care about the cell apoptosis in your story. May it help you and best wishes.
[/quote]
Thanks for the answers. I am currently working with dsRed instead of GFP . Does anyone have experience about PFA fixation on dsRed expressing cells??
Wow, thanks for your explanation but the point is why single cells only?? My boss thinks that aggregrates have to be analyzed too. Because if cells in aggregates are efficiently transfected and knowing that aggregates of our cells can easily be destroyed by pipetting, these cells might be very precious for next steps.
... other opitnion???
#5
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Posted 31 August 2010 - 06:19 AM
Hi!
I am new also in FACS analysis and I will use dsRED plasmid for normalization or transfection efficiency from Clontech.
I tried several times to optimize the transfection efficiency of dsRED plasmid.
For this purpose I transfected the cells the ratio of 1/3 (plasmid/lipofectamin) and after 48 hours transfection, I tripsinized the cells and analysed using BD FACS aria II through PE filter. I checked the exitation and emission for PE. The exitation and emission spectrum was suitable for detection of dsRED. When I analysed the transfection efficiency, I found the ratio about %100. I thought that there may be a problem. I dont know, is this ratio normal? or is this problem?
Thanks for help:)
I am new also in FACS analysis and I will use dsRED plasmid for normalization or transfection efficiency from Clontech.
I tried several times to optimize the transfection efficiency of dsRED plasmid.
For this purpose I transfected the cells the ratio of 1/3 (plasmid/lipofectamin) and after 48 hours transfection, I tripsinized the cells and analysed using BD FACS aria II through PE filter. I checked the exitation and emission for PE. The exitation and emission spectrum was suitable for detection of dsRED. When I analysed the transfection efficiency, I found the ratio about %100. I thought that there may be a problem. I dont know, is this ratio normal? or is this problem?
Thanks for help:)
