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

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.