Talk:20.109(F13): Mod 1 Day 7 FACS analysis: Difference between revisions

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*'''Difference between T/R and W/F samples'''
*'''Difference between T/R and W/F samples'''
**On T/R, plain media wells were exchanged to fresh media, while wells with DMSO or C401 just stayed in the same media.  
**On T/R, plain media wells were exchanged to fresh media, while wells with DMSO or C401 just stayed in the same media that had been added to the plate at 10am on the morning of M1D6.  
**On W/F, all three types of media were available and exchanged to fresh before transfection.
**On W/F, all three types of media were available and exchanged to fresh before transfection.
*'''Collection time consideration'''
*'''Collection time consideration'''
Line 86: Line 86:
|Ratio condition
|Ratio condition
|-
|-
|
|Purple
|
|0.00
|
|0.00
|
|0.00
|
|67.0; 68.6
|
|60.9; 61.3
|
|3.91; 3.39
|
|11.3; 13.8
|
|9.94; 8.90
|
|6.86; 7.34
|
|11.2; 14.9
|
|1:2; 0.1ug D5 and 0.2ug CUT D3
|-
|-
|
|Platinum
|
|0.01
|
|0.00
|
|0.01
|
|68.9; 68.8
|
|69.6; 70.5
|
|4.02; 3.72
|
|11.2; 9.98
|
|8.56; 7.57
|
|4.24; 5.45
|
|14.74; 13.93
|
|1:3; 0.1ug D5 and 0.3ug cut D3
|-
|-
|
|Yellow
|
|0.00
|
|0.00
|
|0.00
|
|71.61; 73.35
|
|76.07; 76.07
|
|4.06; 2.93
|
|13.30; 11.93
|
|11.28; 9.26
|
|2.46; Lost Sample
|
|15.00; 12.78
|
|1:3; 0.1µg ∆5 and 0.3µg cut ∆3
|-
|-
|
|White
|
|0.00
|
|0.00
|
|0.00
|
|76.57; 77.87
|
|76.82; 77.56
|
|3.14; 2.71
|
|7.36; 7.19
|
|6.32; 5.21
|
|3.15; 3.36
|
|8.24; 7.86
|
|1:3; 0.05µg ∆5 and 0.15µg cut ∆3
|-
|-
|
|Pink
|
|0.00
|
|0.02
|
|0.00
|
|55.70; 55.02
|
|64.04; 65.66
|
|3.47; 2.41
|
|5.22; 4.72
|
|3.59; 2.78
|
|1.43; 1.81
|
|2.64; 1.24
|
|4:1, 0.2µg ∆5 and 0.05µg digested ∆3
|-
|-
|
|Orange
|
|0.00
|
|0.02
|
|0.00
|
|71.06; 75.39
|
|69.00; 74.31
|
|2.87; 2.03
|
|11.41; 11.93
|
|9.77; 9.56
|
|7.49; 7.09
|
|17.03; 15.42
|
|4:1, 0.4 µg D5 and 0.1 µg CUT D3
|-
|-
|
|Green
|
|0.00
|
|0.00
|
|0.00
|
|71.12,71.19
|
|64.79,70.18
|
|2.51,2.26
|
|8.98,8.48
|
|7.66,5.86
|
|3.59,2.63
|
|12.10,12.02
|
|1:5, 0.1µg ∆5 and 0.5µg digested ∆3
|-
|-
|
|Blue
|
|0.00
|
|0.00
|
|0.00
|
|48.0, 53.4
|
|51.11, 57.83
|
|2.02, 1.87
|
|10.63, 8.88
|
|7.89, 7.80
|
|3.88, 4.16
|
|1.83, 1.71
|
|1:4 0.1µg ∆5 and 0.4µg digested ∆3
|-
|-
|
|Red
|
|0.00
|
|0.00
|
|0.00
|
|65.17, 66.74
|
|60.46, 63.72
|
|2.47, 2.80
|
|7.50, 8.51
|
|6.85, 6.36
|
|2.74, 3.72
|
|14.00, 16.63
|
|4:1 0.4µg ∆5 and 0.1µg digested ∆3
|-
|-
|}
|}


If you did not collect the default amount of R1 cells (X) per sample, please list your cell count below:
If you did not collect the default amount of R1 cells (10,000) per sample, please list your cell count below:
*Team Example: 5000  
*Team Example: 5000  
*
*


Team Platinum observations: Compared to cells transfected with delta 5 & uncut delta 3, there was more fluorescence in the delta 5 & cut delta 3 sample, and even more in our ratio test of 1 delta 5: 3 delta 3. Compared to the delta 5, delta 3, & DMSO sample, there was less fluorescence in the sample with inhibitor.


Team Yellow observations: There was a significant increase in EGFP fluorescence between ∆5 & cut ∆3 (about 12%) versus ∆5 & uncut ∆3 (about %2). For the samples of ratio 1:3, ∆5:cut ∆3, we observed a higher amount of fluorescence than in the 1:1 experimental samples, although there was some variability between the duplicates. DMSO by itself as a solvent did not seem to have an effect on the fluorescence, but the sample with ∆5, cut ∆3, DMSO + C401 inhibitor had lower EGFP fluorescence than the equivalent samples without inhibitor.
(Teams Platinum and Yellow, your day-of observations are noted -- thanks for posting! Am deleting now for space.)
*Pink Team Observations: There was something wrong with our digested ∆3-plasmid, because we have a smaller percentage of fluoroscence compared to other teams.
W/F Purple team Note: For our unequal ratio test, we forgot to add the diluted lipofectamine to this sample when the rest of the samples received lipofectamine. We added the 50 ul and let it incubate for as long as possible, but that was only about 30 seconds before we had to finish the experiment and clean up to get to our next section.
|


====W/F====
====W/F====
Line 229: Line 231:
|Ratio condition
|Ratio condition
|-
|-
|
|Yellow
|
|0.0
|
|0.0
|
|0.0
|
|66.0; 63.49
|
|53.66; 57.27
|
|0.51; 0.47
|
|12.04; 10.01
|
|5.26; 10.37
|
|8.02; 6.54
|
|0.67; 0.76
|
|10:1 ; 0.02 ug Delta 5, 0.2 ug Cut-Delta 3
|-
|-
|
|Pink
|
|0.0
|
|0.0
|
|0.0
|
|71.3; 66.4
|
|71.9; 72.8
|
|2.5; 2.8
|
|6.4; 6.2
|
|5.1; 5.5
|
|3.5; 2.8
|
|7.2; 5.8
|
|10:1 ; 5ug cut-del3, 0.5ug del5
|-
|-
|
|Red
|
|0.0
|
|0.0
|
|0.0
|
|0.0; 0.0 (Our GFP solution didn't include carrier)
|
|0.0; 0.0
|
|2.88; 2.28
|
|12.48; 10.32
|
|12.24; 11.26
|
|6.66; 5.48
|
|6.92; 8.43
|
|1:5 0.1ug D5 and 0.5ug cut D3
|-
|-
|
|Purple
|
|0.0
|
|0.01
|
|0.0
|
|57.7; 61.5
|
|62.3; 52.7
|
|2.42; 1.78
|
|8.54; 7.81
|
|9.01; 7.09
|
|5.35; 6.33
|
|4.30; 1.33
|
|1:5 .025 ug of Δ5 and .125 ug of digested Δ3
|-
|-
|
|Green
|
|0.0
|
|0.0
|
|0.0
|
|51.54; 56.23
|
|66.78; 59.49
|
|2.46; 2.48
|
|5.33; 4.30
|
|6.10; 4.51
|
|3.77; 4.40
|
|2.44; 18.28
|
|10:1; .5 ug D5: .05 ug digested D3
|-
|-
|
|Blue
|
|0.0
|
|0.0
|
|0.0
|
|59.7; 68.0
|
|63.5; 58.9
|
|1.9; 1.5; 2.4
|
|9.4; 9.4
|
|12.0
|
|7.0; 5.9
|
|14.5; 15.0
|
|5:1; 0.5 µg D5: 0.1 µg digested D3
|-
|-
|
|Orange
|
|0.0
|
|0.0
|
|0.0
|
|66.9; 63.4
|
|70.2; 68.5
|
|2.85; 2.2
|
|9.2; 7.2
|
|11.8; 11.01
|
|6.84; 6.47
|
|14.2; 12.54
|
|10:1 .1 ug D5: .01 ug of D3
|-
|-
|
|White
|
|0.0
|
|0.0
|
|0.0
|
|61.21; 62.41
|
|60.17; 61.49
|
|2.53; 2.56
|
|8.53; 8.43
|
|9.17; 7.07
|
|5.71; 6.73
|
|10.99; 10.66
|
|1:2; 0.1 ug D5: 0.2 ug digested D3
|-
|-
|}
|}


If you did not collect the default amount of R1 cells (X) per sample, please list your cell count below:
If you did not collect the default amount of R1 cells (10,000) per sample, please list your cell count below:
*Team Example: 5000  
*Team Example: 5000  
*
*Green: 4016 (sample 3, D3); 41 (sample 16, 1st ratio test sample), 4163 (sample 17, 2nd ratio test sample)
*Blue: Collected 3 samples of D5+D3, because we had leftover D5+D3 solution so we plated an extra well of it. Only collected one sample of D5+cut D3+DMSO because we forgot to add more media when we were changing the media before adding the appropriate samples, so the cells died.

Latest revision as of 14:01, 9 October 2013

Schedule

Sign up for a time to do flow cytometry in the table below. Please arrive at the time you choose and then expect to spend about 45 minutes in TC and another 45 minutes at FACS. TWO or THREE teams should sign up for each time slot.

T/R

Time block Team 1 Team 2 Team 3
I: 1-2:30 pm Blue Platinum White
II: 2-3:30-pm Purple Yellow Green
III: 3-4:30 pm Orange Pink Red

W/F

Time block Team 1 Team 2 (Team 3)
I: 1-2:30 pm Red Team Orange Team White Team
II: 2-3:30-pm Pink Team Blue Team
III: 3-4:30 pm Purple Team Green Team

Data Summary

Key general notes about experiment

  • Difference between T/R and W/F samples
    • On T/R, plain media wells were exchanged to fresh media, while wells with DMSO or C401 just stayed in the same media that had been added to the plate at 10am on the morning of M1D6.
    • On W/F, all three types of media were available and exchanged to fresh before transfection.
  • Collection time consideration
    • As discussed with most groups, we collected at a high flowrate in order to test many samples.
    • The downside is that transient fluctuations in the data can have a big influence, and may not be averaged out.
    • Keep this trade-off in mind as you look at personal duplicates as well as class-wide data.

Reported data

To aid class-wide comparisons (and statistics!), please post the requested information in the table below in a timely fashion using three significant digits (ex. 3.72). For samples done in duplicate, please separate the two values with a semi-colon. Once you've completed your statistics, don't get over-excited with the number of significant figures you report! For example, "3.7" will suffice. Please describe the ratio test condition in the form "0.2 ug D5 and 0.05 ug CUT D3."

T/R

Team color % R2 gated mock " delta 5 (D5) " delta 3 (D3) " EGFP " EGFP + DMSO " D5 + D3 " D5 + cut D3 " D5 + cut D3 + DMSO " D5 + cut D3 + C401 % R2 Ratio test Ratio condition
Purple 0.00 0.00 0.00 67.0; 68.6 60.9; 61.3 3.91; 3.39 11.3; 13.8 9.94; 8.90 6.86; 7.34 11.2; 14.9 1:2; 0.1ug D5 and 0.2ug CUT D3
Platinum 0.01 0.00 0.01 68.9; 68.8 69.6; 70.5 4.02; 3.72 11.2; 9.98 8.56; 7.57 4.24; 5.45 14.74; 13.93 1:3; 0.1ug D5 and 0.3ug cut D3
Yellow 0.00 0.00 0.00 71.61; 73.35 76.07; 76.07 4.06; 2.93 13.30; 11.93 11.28; 9.26 2.46; Lost Sample 15.00; 12.78 1:3; 0.1µg ∆5 and 0.3µg cut ∆3
White 0.00 0.00 0.00 76.57; 77.87 76.82; 77.56 3.14; 2.71 7.36; 7.19 6.32; 5.21 3.15; 3.36 8.24; 7.86 1:3; 0.05µg ∆5 and 0.15µg cut ∆3
Pink 0.00 0.02 0.00 55.70; 55.02 64.04; 65.66 3.47; 2.41 5.22; 4.72 3.59; 2.78 1.43; 1.81 2.64; 1.24 4:1, 0.2µg ∆5 and 0.05µg digested ∆3
Orange 0.00 0.02 0.00 71.06; 75.39 69.00; 74.31 2.87; 2.03 11.41; 11.93 9.77; 9.56 7.49; 7.09 17.03; 15.42 4:1, 0.4 µg D5 and 0.1 µg CUT D3
Green 0.00 0.00 0.00 71.12,71.19 64.79,70.18 2.51,2.26 8.98,8.48 7.66,5.86 3.59,2.63 12.10,12.02 1:5, 0.1µg ∆5 and 0.5µg digested ∆3
Blue 0.00 0.00 0.00 48.0, 53.4 51.11, 57.83 2.02, 1.87 10.63, 8.88 7.89, 7.80 3.88, 4.16 1.83, 1.71 1:4 0.1µg ∆5 and 0.4µg digested ∆3
Red 0.00 0.00 0.00 65.17, 66.74 60.46, 63.72 2.47, 2.80 7.50, 8.51 6.85, 6.36 2.74, 3.72 14.00, 16.63 4:1 0.4µg ∆5 and 0.1µg digested ∆3

If you did not collect the default amount of R1 cells (10,000) per sample, please list your cell count below:

  • Team Example: 5000


(Teams Platinum and Yellow, your day-of observations are noted -- thanks for posting! Am deleting now for space.)

  • Pink Team Observations: There was something wrong with our digested ∆3-plasmid, because we have a smaller percentage of fluoroscence compared to other teams.

W/F Purple team Note: For our unequal ratio test, we forgot to add the diluted lipofectamine to this sample when the rest of the samples received lipofectamine. We added the 50 ul and let it incubate for as long as possible, but that was only about 30 seconds before we had to finish the experiment and clean up to get to our next section. |

W/F

Team color % R2 gated mock " delta 5 (D5) " delta 3 (D3) " EGFP " EGFP + DMSO " D5 + D3 " D5 + cut D3 " D5 + cut D3 + DMSO " D5 + cut D3 + C401 % R2 Ratio test Ratio condition
Yellow 0.0 0.0 0.0 66.0; 63.49 53.66; 57.27 0.51; 0.47 12.04; 10.01 5.26; 10.37 8.02; 6.54 0.67; 0.76 10:1 ; 0.02 ug Delta 5, 0.2 ug Cut-Delta 3
Pink 0.0 0.0 0.0 71.3; 66.4 71.9; 72.8 2.5; 2.8 6.4; 6.2 5.1; 5.5 3.5; 2.8 7.2; 5.8 10:1 ; 5ug cut-del3, 0.5ug del5
Red 0.0 0.0 0.0 0.0; 0.0 (Our GFP solution didn't include carrier) 0.0; 0.0 2.88; 2.28 12.48; 10.32 12.24; 11.26 6.66; 5.48 6.92; 8.43 1:5 0.1ug D5 and 0.5ug cut D3
Purple 0.0 0.01 0.0 57.7; 61.5 62.3; 52.7 2.42; 1.78 8.54; 7.81 9.01; 7.09 5.35; 6.33 4.30; 1.33 1:5 .025 ug of Δ5 and .125 ug of digested Δ3
Green 0.0 0.0 0.0 51.54; 56.23 66.78; 59.49 2.46; 2.48 5.33; 4.30 6.10; 4.51 3.77; 4.40 2.44; 18.28 10:1; .5 ug D5: .05 ug digested D3
Blue 0.0 0.0 0.0 59.7; 68.0 63.5; 58.9 1.9; 1.5; 2.4 9.4; 9.4 12.0 7.0; 5.9 14.5; 15.0 5:1; 0.5 µg D5: 0.1 µg digested D3
Orange 0.0 0.0 0.0 66.9; 63.4 70.2; 68.5 2.85; 2.2 9.2; 7.2 11.8; 11.01 6.84; 6.47 14.2; 12.54 10:1 .1 ug D5: .01 ug of D3
White 0.0 0.0 0.0 61.21; 62.41 60.17; 61.49 2.53; 2.56 8.53; 8.43 9.17; 7.07 5.71; 6.73 10.99; 10.66 1:2; 0.1 ug D5: 0.2 ug digested D3

If you did not collect the default amount of R1 cells (10,000) per sample, please list your cell count below:

  • Team Example: 5000
  • Green: 4016 (sample 3, D3); 41 (sample 16, 1st ratio test sample), 4163 (sample 17, 2nd ratio test sample)
  • Blue: Collected 3 samples of D5+D3, because we had leftover D5+D3 solution so we plated an extra well of it. Only collected one sample of D5+cut D3+DMSO because we forgot to add more media when we were changing the media before adding the appropriate samples, so the cells died.
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