We diluted 100µL of cell culture into 400 µL of Phosphate Buffered Saline (PBS). For our induction experiments, we split and induced the cells with IPTG 6 hours after they were inoculated. Four hours after the cells were induced, we diluted the cells in order to obtain an ideal concentration of OD600 0.01. The cells were then grown for another five hours and taken out every 20 minutes to be filtered on ice and FACSed immediately. To acquire data with flow cytometry, we adjust side-scatter (SSC) and
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The procedure for FACS time course experiments was as follows: Cells were inoculated from LB agar plates in the morning into fresh M9 containing appropriate selective antibiotics. They were then grown for between 7-10 hours in a 37C shaking incubator at 250 rpm. Then, OD600 was measured using a spectrophotometer, and cells were diluted to an OD600 of .01 into fresh inducer containing media. Time points were collected directly onto ice, and the time out of the shaking incubator for cells was kept to less than 2 minutes. Iced time points were then immediatly FACSed, and controls were performed to ensure cells were not changing fluorescence over time. Cells were diluted every 100 minutes into fresh pre-warmed M9 to maintain midlog growth. </div>
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forward scatter (FSC) PMT voltages using bacteria from one of our samples until the distribution of each
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is centered on the scale and we adjust FITC/GFP PMT voltage until the upper edge of the “bell curve”
from the fluorescent population is one order of magnitude below the upper end of the scale. We run the
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Flow cytometry data was aquired on the FL1 (sfGFP/MEFL) or FL3 (mScarlet-I/RFP/MECY) channel, with samples being kept at 4C throughout. Voltages and gatings were standardized from early on, and were always set to the same settings (FSC/SSC: 500, FL1 gain: 600, FL3 gain: 675).
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program enough to acquire at least 10,000 events for each biological sample. After running the samples,
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At least 10,000 (typically 20,000) events/cells collected after gating were collected from each biological replicate, and at least 20,000 events were collected each day from absolute fluorescence calibration beads (SpheroTech Rainbow Calibration Particles RCP-30-5A, bead lots AH01, AB01) to allow for setting independent conversion to absolute units.
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we acquire at least 10,000 events from a sample of calibration beads with the voltages for FSC at 339
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FlowCal was used for downstream analysis and conversions to absolute fluorescence units. The two
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and SSC at 292 and at the voltages applied for the sample collection on the channel(s) used. We used
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absolute fluorescence units we used are Molecules of Equivalent Fluorescein (MEFL) for GFP and Molecules of Equivalent CY5 (MECY) for mScarlet-I.</div>
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SpheroTech Rainbow Calibration Particles RCP-30-5A for fluorescent calibration beads because they
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have been calibrated for excitation and detection of the particles in most channels of any flow
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cytometer. Every 100 minutes, we diluted the cells 1:10 in pre-warmed culture. We used FlowCal for downstream analysis and conversion to absolute fluorescence units. The two
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absolute fluorescence units we used are Molecules of Equivalent Fluorescein (MEFL) for GFP and MEPTR for mScarlet.</div
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Revision as of 18:35, 1 November 2017
Experimental Protocol
Flow Cytometry Time Courses
All time course measurements were carried out in the E. coli strain NEB 10-Beta (ΔAra-Leu) in M9 media with .1% casamino acids, .4% glucose (or glycerol when arabinose was used), and 500mM L-leucine, and were measured using a BioRad S3e Cell Sorter, with a minimum of 10,000 cells measured from each of three biological replicates (colonies) collected on the same day. Care was made to maintain cells in midlog growth phase.
The procedure for FACS time course experiments was as follows: Cells were inoculated from LB agar plates in the morning into fresh M9 containing appropriate selective antibiotics. They were then grown for between 7-10 hours in a 37C shaking incubator at 250 rpm. Then, OD600 was measured using a spectrophotometer, and cells were diluted to an OD600 of .01 into fresh inducer containing media. Time points were collected directly onto ice, and the time out of the shaking incubator for cells was kept to less than 2 minutes. Iced time points were then immediatly FACSed, and controls were performed to ensure cells were not changing fluorescence over time. Cells were diluted every 100 minutes into fresh pre-warmed M9 to maintain midlog growth.
Flow cytometry data was aquired on the FL1 (sfGFP/MEFL) or FL3 (mScarlet-I/RFP/MECY) channel, with samples being kept at 4C throughout. Voltages and gatings were standardized from early on, and were always set to the same settings (FSC/SSC: 500, FL1 gain: 600, FL3 gain: 675).
At least 10,000 (typically 20,000) events/cells collected after gating were collected from each biological replicate, and at least 20,000 events were collected each day from absolute fluorescence calibration beads (SpheroTech Rainbow Calibration Particles RCP-30-5A, bead lots AH01, AB01) to allow for setting independent conversion to absolute units.
FlowCal was used for downstream analysis and conversions to absolute fluorescence units. The two
absolute fluorescence units we used are Molecules of Equivalent Fluorescein (MEFL) for GFP and Molecules of Equivalent CY5 (MECY) for mScarlet-I.
