SCYM Cell Sorting 5 — Questions and Answers
Question 1: What is the significance of 'satellite droplets' in a droplet-based cell sorter?
- They carry extra charge to enhance deflection accuracy
- They are small droplets that can carry cells and contaminate adjacent sort streams (Correct answer)
- They improve breakoff point stability at high event rates
- They are used to calibrate the sort delay
Correct answer: They are small droplets that can carry cells and contaminate adjacent sort streams
Satellite droplets are tiny droplets that form alongside the main droplets and can carry cells into adjacent collection streams, potentially contaminating sorted populations.
Question 2: A researcher sorting GFP-expressing cells notices that the sorted GFP-positive fraction contains many GFP-negative cells. Which instrument parameter should be investigated first?
- Laser power
- Sort delay (drop delay) accuracy (Correct answer)
- PMT gain on the GFP channel
- Sheath fluid conductivity
Correct answer: Sort delay (drop delay) accuracy
When gated purity is adequate but sorted purity is low, an incorrect sort delay is the most common cause, as it means the wrong droplet is being charged and deflected.
Question 3: Which aerosol containment strategy is most effective for sorting infectious or BSL-2 human samples on a conventional jet-in-air sorter?
- Running the sort at reduced flow rate to lower event speed
- Using an enclosed sort chamber with HEPA-filtered exhaust and working under negative pressure (Correct answer)
- Pre-fixing the sample with 1% paraformaldehyde before sorting
- Sorting in an open biosafety cabinet
Correct answer: Using an enclosed sort chamber with HEPA-filtered exhaust and working under negative pressure
An enclosed sort chamber with HEPA-filtered exhaust and negative pressure prevents aerosols containing potentially infectious particles from escaping into the operator's environment.
Question 4: What does a high event rate on the sorter primarily increase the risk of, even when sample concentration appears low?
- PMT photobleaching
- Coincident events leading to reduced sort purity (Correct answer)
- Sheath fluid contamination
- Laser beam divergence
Correct answer: Coincident events leading to reduced sort purity
High event rates increase the probability that two cells arrive at the interrogation point within the same droplet window, causing coincident events that reduce sort purity.
Question 5: When sorting for downstream single-cell RNA sequencing (scRNA-seq), why is it important to minimize the time cells spend at room temperature during sorting?
- Warm temperatures cause fluorescent dyes to quench
- Ex vivo time at room temperature alters transcriptional profiles, introducing artifacts (Correct answer)
- Room temperature reduces droplet formation stability
- Ambient temperature increases nonspecific antibody binding
Correct answer: Ex vivo time at room temperature alters transcriptional profiles, introducing artifacts
Cells rapidly change their gene expression profiles in response to stress and environmental changes, so minimizing pre-sort time at room temperature helps preserve in vivo transcriptional states.
Question 6: Which approach is best for maintaining cell viability when sorting must take more than 2 hours?
- Adding DMSO to the sample at 5% concentration
- Keeping the sample tube chilled on ice or in a refrigerated sample chamber throughout the sort (Correct answer)
- Increasing sheath pressure to speed up the sort
- Adding propidium iodide to the sample before sorting
Correct answer: Keeping the sample tube chilled on ice or in a refrigerated sample chamber throughout the sort
Cooling the sample to 4°C slows metabolic activity and reduces stress-induced cell death, making it the standard practice for long sorting sessions.
Question 7: In a four-way sort, cells are deflected into four separate collection streams. What instrument feature makes this possible?
- Four independent lasers, one per sort stream
- Two pairs of high-voltage deflection plates oriented at different voltages to create four distinct deflection angles (Correct answer)
- Four separate sample injection nozzles running simultaneously
- Four PMT detectors each linked to a dedicated deflection circuit
Correct answer: Two pairs of high-voltage deflection plates oriented at different voltages to create four distinct deflection angles
Four-way sorting uses two pairs of deflection plates with independent voltage levels, creating four distinct deflection trajectories (left high, left low, right high, right low) to direct droplets into four tubes.
SCYM Cell Sorting 5
What is the significance of 'satellite droplets' in a droplet-based cell sorter?