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Cell Sorting Protocol

Fluorescence-activated cell sorting (FACS) is used to isolate single E. coli transformed with the assembled library into individual wells of 384-well plates.

Overview

Key advantages of FACS for parsing clone:

  • Accessibility: FACS core facilities are widely available at most research institutions
  • Speed: Standard FACS machines (BD FACSAria II, Sony SH800) sort one 384-well plate in 5-8 minutes
  • Scalability: Isolate thousands of clones per day
  • Generalizability: FSC/SSC gating is sufficient to isolate cells, obviating the need for a fluorescent marker

Materials

Item Supplier Notes
384-well plates Thermo Fisher (242757) Black-walled, clear flat bottom
Breathable seals VWR (60941-086) Allows gas exchange during growth
LB + antibiotic — 50 µL per well
Flow cytometer — With 96- or 384-well plate sorting capability
Sterile PBS — For dilution and sheath fluid

Protocol

1. Prepare Cells for Sorting

  1. Inoculate 5 mL LB + antibiotic with glycerol stock from library assembly
  2. Grow overnight at 37 °C with shaking (250 rpm)
  3. Dilute overnight culture 1:10 in PBS (100 µL of culture in 1 mL buffer)
  4. Pellet at 5,000 × g for 5 min
  5. Resuspend pellet in 1 mL sterile PBS
  6. Repeat pellet and wash step once more
  7. Dilute to 1:2 in PBS for sorting (final volume should be ≥ 1 mL)

2. Prepare 384-Well Plates

  1. Add 40 µL LB + antibiotic to each well using a multichannel pipette or liquid handler
  2. Seal plates
  3. Keep at room temperature until ready to sort

3. Configure Flow Cytometer

Recommended gating strategy:

  • FSC vs. SSC: Gate on bacterial population (remove debris and doublets)
  • Single-cell mode: Sort exactly 1 event per well
  • Purity mode: Use highest purity setting to minimize contamination
  • [To-do]: [Add full FACS protocol details]

Typical sort parameters:

Parameter Value
Nozzle size 100 µm
Sheath pressure 20 psi
Sort mode Single cell (1 drop, 0 phase)
Events per well 1
Target wells All 384 wells
Sort rate ~500 events/sec

4. Perform Sorting

  1. Load cell suspension into flow cytometer
  2. Set FSC/SSC gates to capture live bacterial population
  3. Load 384-well plate onto sorting stage
  4. Run calibration/alignment if needed
  5. Begin sort: 1 cell per well across all 384 wells
  6. Remove plate when complete, seal with foil or plastic cover
  7. Repeat steps 5 and 6 for additional plates
💡 Tip: Ensure gating excludes debris (low FSC/SSC) and doublets.

5. Outgrowth

  1. Incubate sorted plates at 37 °C for 12–16 hours (overnight)
  2. Check for growth by visual inspection or OD600 measurement
  3. Expect 60–75% of wells to show growth (typical for single-cell sorts)

Determining Sort Scale

The number of wells to sort depends on your library size and desired coverage. Use usortm estimate to calculate the recommended oversampling: 

# For a 500-variant library with default 4x oversampling
usortm estimate --library-size 500

# For a skewed library, increase oversampling
usortm estimate --library-size 500 --fold-sampling 8 --skew 10

General guidelines:

Library Size Fold Sampling Total Wells Number of Plates
100 4× 400 2
500 4× 2,000 6
1,000 4× 4,000 11
2,000 6× 12,000 32

Quality Control

Troubleshooting

Problem Possible Cause Solution
Low growth rate (<50%) Cells stressed during sort Keep cells on ice, reduce sort time, use fresher culture
Very high growth rate (>90%) Multiple cells per well Check single-cell gating, reduce cell concentration
Clogged nozzle Cell aggregates or debris Filter cell suspension through 40 µm cell strainer
Slow sort rate Cell concentration too low Increase cell concentration to 106–107 cells/mL
Uneven growth across plate Edge effects, evaporation Use breathable seals, humidified incubator, avoid outer wells

Next Steps

After overnight growth, proceed to PCR barcoding to add well-specific DNA identifiers to each clone.