SCYM Cell Sorting 2 — Questions and Answers
Question 1: What is the primary purpose of a sheath fluid in a fluorescence-activated cell sorter (FACS)?
- To lyse red blood cells before sorting
- To hydrodynamically focus cells into a single-file stream through the interrogation point (Correct answer)
- To provide nutrients to maintain cell viability during sorting
- To prevent photobleaching of fluorescent dyes
Correct answer: To hydrodynamically focus cells into a single-file stream through the interrogation point
Sheath fluid surrounds the sample core and uses laminar flow to hydrodynamically focus cells into a single-file stream at the laser interrogation point.
Question 2: In electrostatic cell sorting, a droplet containing a cell of interest receives a charge. What happens next?
- The droplet is heated to lyse non-target cells
- The charged droplet is deflected by high-voltage deflection plates into a collection vessel (Correct answer)
- The droplet is UV-irradiated to confirm cell identity
- The droplet is recirculated for a second sort pass
Correct answer: The charged droplet is deflected by high-voltage deflection plates into a collection vessel
Charged droplets are deflected by high-voltage electrostatic plates, directing target cells into the appropriate collection tube while uncharged droplets go to waste.
Question 3: Which sort mode should be selected when recovering the purest possible population is the top priority, even at the cost of yield?
- Yield mode
- Purity mode (Correct answer)
- Enrich mode
- Single-cell mode
Correct answer: Purity mode
Purity mode aborts the sort decision if a coincident event is detected, ensuring only unambiguously identified target cells are collected.
Question 4: What does the term 'coincidence' refer to in the context of cell sorting?
- Two lasers firing at the same target cell simultaneously
- Two or more cells occupying the same droplet at the time of deflection decision (Correct answer)
- A cell expressing two different fluorescent markers
- A sort event that matches the gating strategy perfectly
Correct answer: Two or more cells occupying the same droplet at the time of deflection decision
Coincidence occurs when more than one cell is present in a single droplet, making it impossible to sort the target cell without co-sorting an unwanted cell.
Question 5: The 'abort rate' reported during a sort session primarily reflects which condition?
- Events excluded by doublet discrimination gating
- Sort decisions cancelled due to coincident events near or in the same droplet (Correct answer)
- The percentage of laser power not absorbed by cells
- Events lost due to PMT saturation
Correct answer: Sort decisions cancelled due to coincident events near or in the same droplet
The abort rate represents the frequency at which sort decisions are cancelled because a coincident event was detected that would compromise purity.
Question 6: Why is it important to keep the sample concentration low when setting up a high-purity sort?
- High concentrations cause clogging of the nozzle tip
- Lower concentrations reduce coincident events, improving sort purity (Correct answer)
- High concentrations deplete sheath fluid faster
- Low concentrations allow more laser photons per cell
Correct answer: Lower concentrations reduce coincident events, improving sort purity
Lower sample concentrations reduce the probability of two cells occupying the same droplet simultaneously, thereby minimizing coincidence and improving purity.
Question 7: A researcher sorts cells into a 96-well plate. Which sort mode is most appropriate for depositing exactly one cell per well?
- Purity mode
- Yield mode
- Single-cell mode (Correct answer)
- Bulk sort mode
Correct answer: Single-cell mode
Single-cell mode is specifically designed to ensure that exactly one target cell is deposited per sort event, critical for cloning or single-cell genomics applications.
SCYM Cell Sorting 2
What is the primary purpose of a sheath fluid in a fluorescence-activated cell sorter (FACS)?