SCYM Cell Sorting 4 — Questions and Answers
Question 1: What is the primary advantage of microfluidic (chip-based) cell sorting compared to conventional droplet-based FACS?
- Higher throughput exceeding 100,000 events per second
- A closed, contained fluidic path that reduces aerosol biohazard risk (Correct answer)
- Superior sensitivity for detecting dim fluorescent signals
- Ability to sort cells based on size alone without fluorescent labels
Correct answer: A closed, contained fluidic path that reduces aerosol biohazard risk
Chip-based microfluidic sorters keep the sample in a sealed cartridge, dramatically reducing aerosol generation and making them safer for sorting BSL-2 or BSL-3 samples.
Question 2: Which type of sort purity verification is recommended immediately after collecting a sorted fraction?
- Re-analyzing a portion of the sorted sample on the same or a validated cytometer (Correct answer)
- Sequencing the sorted cells to confirm surface marker expression
- Staining sorted cells with Trypan blue and counting on a hemocytometer
- Culturing sorted cells for 24 hours before any assessment
Correct answer: Re-analyzing a portion of the sorted sample on the same or a validated cytometer
Re-analyzing a portion of the sorted fraction on the cytometer is the standard method to verify that the collected cells match the target gate.
Question 3: In a clinical or GMP-compliant cell sorting environment, which factor is most critical to document for regulatory purposes?
- The brand of sheath fluid used
- Chain of custody, sort parameters, sterility testing, and instrument QC logs (Correct answer)
- The fluorochrome lot numbers used for staining
- The ambient room temperature during the sort
Correct answer: Chain of custody, sort parameters, sterility testing, and instrument QC logs
GMP sorting requires comprehensive documentation including chain of custody records, validated instrument QC, sort parameters, and sterility testing to satisfy regulatory requirements.
Question 4: What is the main reason to use a low-pressure (low-PSI) nozzle when sorting fragile primary cells?
- Low pressure increases droplet charge intensity
- Lower pressure reduces shear forces, improving post-sort cell viability (Correct answer)
- Low pressure slows the event rate, allowing more accurate gating
- Lower pressure reduces laser scatter background
Correct answer: Lower pressure reduces shear forces, improving post-sort cell viability
Fragile cells, such as neurons or activated T cells, are susceptible to mechanical damage from high shear forces; lower pressure nozzles reduce these forces and improve viability.
Question 5: When sorting rare populations (less than 0.1% of events), which strategy best improves efficiency?
- Increasing laser power to improve fluorescence signal
- Pre-enriching the sample using magnetic bead selection before FACS sorting (Correct answer)
- Switching to a larger nozzle to increase flow rate
- Reducing PMT voltages to prevent saturation
Correct answer: Pre-enriching the sample using magnetic bead selection before FACS sorting
Pre-enriching the sample with magnetic beads increases the frequency of the target population before sorting, dramatically reducing sort time and improving recovery.
Question 6: What effect does increasing sample pressure relative to sheath pressure have on the sample core stream?
- It widens the sample core, increasing the chance of two cells being interrogated simultaneously (Correct answer)
- It narrows the sample core, improving focus and reducing interrogation volume
- It has no effect on the core diameter if sheath pressure is constant
- It forces cells to flow faster than the sheath, causing turbulent flow
Correct answer: It widens the sample core, increasing the chance of two cells being interrogated simultaneously
Increasing sample pressure relative to sheath pressure widens the sample core, which increases coincidence rates and can reduce the quality of laser interrogation.
Question 7: Which collection buffer additive is commonly used to prevent sorted lymphocytes from adhering to the collection tube walls?
- EDTA or heparin (Correct answer)
- Formaldehyde
- Saponin
- Propidium iodide
Correct answer: EDTA or heparin
EDTA chelates calcium ions and, along with heparin, reduces cell clumping and adhesion to tube walls by inhibiting integrin-mediated and coagulation-mediated sticking.
SCYM Cell Sorting 4
What is the primary advantage of microfluidic (chip-based) cell sorting compared to conventional droplet-based FACS?