Cell Sorting

Overview of Cell Sorting

Cell Sorting:
- Definition: A technique that uses flow cytometry to identify and physically separate cells based on their fluorescence and scatter properties
- Also Known As: Fluorescence-Activated Cell Sorting (FACS)
Purpose:
- Isolate Specific Cell Populations: To obtain pure populations of cells for downstream applications
- Enrich Rare Cells: To increase the frequency of rare cells for further analysis
- Study Cell Function: To study the function of specific cell populations in vitro or in vivo
Applications:
- Immunology
- Cell Biology
- Stem Cell Research
- Cancer Research
- Genomics

Sample Preparation:
- Prepare a single-cell suspension of the sample
- Label the cells with fluorescent antibodies or dyes
- Filter the sample to remove cell aggregates and debris
Flow Cytometry Analysis:
- Pass the cells through a flow cytometer, where their fluorescence and scatter properties are measured
- Identify the cell populations of interest based on their marker expression
Droplet Formation:
- The flow cytometer creates droplets containing individual cells
- The timing of droplet formation is precisely controlled
Charging:
- Droplets containing cells of interest are given an electrical charge
- The charge can be positive, negative, or neutral, depending on the desired sorting parameters
Deflection:
- Charged droplets are deflected by an electric field into collection tubes
- Neutral droplets are not deflected and are discarded
Collection:
- Sorted cells are collected in tubes for further analysis or culture

Fluidics System:
- Transports the cells from the sample tube to the sorting nozzle
- Maintains a stable and consistent flow rate
Optics System:
- Excites the fluorochromes on the cells
- Collects the emitted fluorescence and scattered light
- Directs the light to the detectors
Electronics System:
- Processes the signals from the detectors
- Identifies the cell populations of interest
- Controls the charging and deflection of the droplets
Sorting Nozzle:
- Forms droplets containing individual cells
- The size of the nozzle affects the size of the droplets and the sorting efficiency
Deflection Plates:
- Create an electric field that deflects the charged droplets into collection tubes
- The voltage applied to the deflection plates affects the degree of deflection
Collection Tubes:
- Collect the sorted cells
- Can be chilled to improve cell viability

Purity Mode:
- Goal: To maximize the purity of the sorted population
- Settings: Uses more stringent sorting criteria
- Tradeoff: May result in lower recovery
Yield Mode:
- Goal: To maximize the recovery of the sorted population
- Settings: Uses less stringent sorting criteria
- Tradeoff: May result in lower purity
Single-Cell Mode:
- Goal: To sort individual cells into separate wells or tubes
- Settings: Requires precise control of droplet formation and charging
- Applications: Cloning, single-cell sequencing

Cell Viability:
- Cell sorting can be stressful for cells, so it is essential to maintain high cell viability
- Use appropriate cell culture techniques and handling procedures
- Use a viability dye to exclude dead cells from the sorting process
Sterility:
- Cell sorting must be performed under sterile conditions to prevent contamination
- Use sterile reagents and supplies
- Clean and disinfect the cell sorter regularly
Carryover:
- Carryover occurs when cells from one sample contaminate subsequent samples
- Minimize carryover by using appropriate washing procedures and by using a dedicated nozzle for each sample type
Compensation:
- Accurate compensation is essential for cell sorting
- Use single-stain controls to accurately calculate compensation values
- Verify compensation settings before sorting
Drop Delay:
- The drop delay is the time it takes for a cell to travel from the laser intercept to the droplet break-off point
- The drop delay must be accurately calibrated to ensure that the correct droplets are charged and deflected

Immunology:
- Isolating specific immune cell populations for functional studies
- Cloning T cell or B cell receptors
- Generating monoclonal antibodies
Cell Biology:
- Isolating cells with specific characteristics (e.g., size, shape, fluorescence intensity)
- Studying cell differentiation and development
- Generating stable cell lines
Stem Cell Research:
- Isolating hematopoietic stem cells (HSCs) for transplantation
- Isolating mesenchymal stem cells (MSCs) for regenerative medicine applications
- Studying stem cell differentiation and self-renewal
Cancer Research:
- Isolating cancer stem cells
- Studying the mechanisms of cancer metastasis
- Developing new cancer therapies
Genomics:
- Preparing samples for single-cell sequencing
- Isolating specific cell populations for gene expression analysis *Identifying genetic markers

Low Cell Recovery:
- Possible Causes:
  - Cell loss during sample preparation
  - Inefficient sorting
  - Incorrect drop delay
- Troubleshooting Steps:
  - Optimize sample preparation protocols
  - Adjust the sorting parameters
  - Calibrate the drop delay
Poor Purity:
- Possible Causes:
  - Incorrect gating
  - Inadequate compensation
  - Cell aggregation
- Troubleshooting Steps:
  - Review gating strategy
  - Verify compensation values
  - Disaggregate cells
Cell Damage:
- Possible Causes:
  - High sorting pressure
  - Prolonged sorting time
  - Inappropriate collection buffer
- Troubleshooting Steps:
  - Reduce sorting pressure
  - Shorten sorting time
  - Use appropriate collection buffer

Cell Sorting (FACS): A technique that uses flow cytometry to identify and physically separate cells
Droplet Formation: The process of creating droplets containing individual cells
Charging: The process of giving droplets containing cells of interest an electrical charge
Deflection: The process of using an electric field to deflect charged droplets into collection tubes
Purity Mode: A sorting mode that maximizes the purity of the sorted population
Yield Mode: A sorting mode that maximizes the recovery of the sorted population
Single-Cell Mode: A sorting mode that sorts individual cells into separate wells or tubes