Part 2: Flow cytometry analysis:
←Older revision
Revision as of 22:54, 2 April 2015
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#The pdf files with your data are posted on the M2D6 Talk page.
#The pdf files with your data are posted on the M2D6 Talk page.
#The instructor samples are listed in the table below. From this table, and from the T/R and W/F image sets, try to address the questions below.
#The instructor samples are listed in the table below. From this table, and from the T/R and W/F image sets, try to address the questions below.
-
#*Background. The scatter data is used – in three steps – to make gate P3, which should consist primarily of
live
,
single
cells. From the cells gated in
P3
, two sub-gates are made that capture all GFP-positive cells ("Green cells" gate) and all BFP-positive cells ("Blue cells" gate). Both singly and doubly positive cells are included in each gate. It is important to read the "% Parent" statistics: these indicate XFP-positive cells as a percentage of all the cells in
P3
. The "% Total" statistics include debris, aggregates, and clearly dead cells!
+
#*Background. The scatter data is used – in three steps – to make gate P3, which should consist primarily
single cells. Next a gate that we called '''Live cells''' was set based upon the addition ToPro3 to the media. ToPro3 can only pass through the membranes
of
dead cells
,
staining them with a dye that fluoresces when exited with 647 nm wavelength light. The cytometer we used calls this channel the '''APC''' channel (because the dye APC also fluoresces at that wavelength). So, using the plot that shows FSC vs APC we can ''gate-in'' the live
cells
(exclude the dead ToPro3+ cells)
.
+
#
From the cells gated in
Live Cells
, two sub-gates are made that capture all GFP-positive cells ("Green cells" gate) and all BFP-positive cells ("Blue cells" gate). Both singly and doubly positive cells are included in each gate. It is important to read the "% Parent" statistics: these indicate XFP-positive cells as a percentage of all the cells in
Live Cells
. The "% Total" statistics include debris, aggregates, and clearly dead cells!
#*What percent Green cells are in the mock sample on each day? What about Blue cells?
#*What percent Green cells are in the mock sample on each day? What about Blue cells?
-
#*What percent of singly-transfected cells express GFP? Do
within-day and
cross-day replicates agree well or not?
+
#*What percent of singly-transfected cells express GFP? Do cross-day replicates agree well or not?
-
#*What percent of singly-transfected cells express BFP? Do
within-day and
cross-day replicates agree well or not?
+
#*What percent of singly-transfected cells express BFP? Do cross-day replicates agree well or not?
-
#*What percent of co-transfected cells express GFP? Express BFP?
Comparing the Green and Blue gates to Q1 and Q4, about what percent of cells seem co-transfected, versus expressing just GFP, and expressing just BFP
?
+
#Now, start to look at your K1-intact conditions (and possibly those of your classmates).
-
#*How is within-day and cross-day replicate agreement for the co-transfected samples?
Do
the
tables below suggest an explanation for why?
+
#*What percent of co-transfected cells express GFP? Express BFP?
How many express both
?
-
#*Does
ethanol
appear to affect scatter profiles? What about affecting
GFP, BFP, or
co-expression?
+
#*How is within-day and cross-day replicate agreement for the co-transfected samples?
-
#*
What NHEJ repair value do
you
calculate for Zac
'
s original BFP plasmid, using
the
first replicate in the W/F instructor data? Try this calculation by hand, using the mean fluorescence intensity. Later, you can include this data as
a
check on your Excel worksheet. The value you should calculate is 12.8%.<font color=red>Update: Your instructor picked off GFP mean fluorescence instead of BFP mean fluorescence for the intact case! Here is where computers definitely beat manual picking off of data. The correct number is '''8.7%'''
.
</font color>
+
#Answer
the
following by looking at a team that used DMNB (hint: there is a spreadsheet on the Talk page that will help you locate those samples).
-
#After you understand the instructor data, skim over your 12 sample plots. Can you see apparent differences between K1, K1+
401
, and xrs6?
+
#*Does
DMSO
appear to affect scatter profiles? What about affecting co-expression?
+
#*
Hint:
you'
ll need to compare
the
K1 intact samples from
a
DMNB group versus a different group
.
+
#After you understand the instructor data, skim over your 12 sample plots. Can you see apparent differences between K1, K1+
inhibitor
, and xrs6?
#Now that you have a good conceptual understanding of the data, it's time to crunch some numbers. Open the .csv file and save it as a newly named .xlsx file.
#Now that you have a good conceptual understanding of the data, it's time to crunch some numbers. Open the .csv file and save it as a newly named .xlsx file.
#Begin by deleting all of the rows except the twelve containing your own dataset.
#Begin by deleting all of the rows except the twelve containing your own dataset.
-
#Next delete all of the columns except the few that interest you. Keep in mind that you need to know Green cell and Blue cell gating as a % of the parent gate,
P3
. Class-wide, you are only required to do your calculations based on mean fluorescence intensity (MFI)
, to be consistent with Samson lab data
.
However, you may find it interesting to see whether using median fluorescence intensity gives you the same trends or not. Just a few extra copy-pastes to do both calculations!
+
#Next delete all of the columns except the few that interest you. Keep in mind that you need to know Green cell and Blue cell gating as a % of the parent gate,
Live Cells
. Class-wide, you are only required to do your calculations based on mean fluorescence intensity (MFI).
#We recommend that you prepare a new Excel file with your NHEJ equations, and just copy-paste in the appropriate % and MFI data; this approach is a versatile one. Your final worksheet might look similar to the screenshot below.
#We recommend that you prepare a new Excel file with your NHEJ equations, and just copy-paste in the appropriate % and MFI data; this approach is a versatile one. Your final worksheet might look similar to the screenshot below.
#Remember that for each of the twelve wells you should calculate raw reporter expressions and a BFP/GFP normalized value. Then, for each intact/cut pair you can calculate an NHEJ value. In this way, we should have quadruplicate NHEJ values for most repair topology/cell population conditions, which will allow us to do statistical comparisons.
#Remember that for each of the twelve wells you should calculate raw reporter expressions and a BFP/GFP normalized value. Then, for each intact/cut pair you can calculate an NHEJ value. In this way, we should have quadruplicate NHEJ values for most repair topology/cell population conditions, which will allow us to do statistical comparisons.
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'''Reference information:'''
'''Reference information:'''
-
[[Image:S14M2_TR-inst-samples.png|thumb|left|300px|'''TR instructor samples.''']]
-
[[Image:S14M2_WF-inst-samples.png|thumb|center|250px|'''WF instructor samples.''']]
<br style="clear:both;"/>
<br style="clear:both;"/>
[[Image:S14M2_worksheet-example.png|thumb|center|550px|'''Sample NHEJ calculator screenshot.''']]
[[Image:S14M2_worksheet-example.png|thumb|center|550px|'''Sample NHEJ calculator screenshot.''']]