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 (242764) Black-walled, transparent flat bottom (required for OD600 reads)
Adhesive foil seals Thermo Fisher (AB0626) Seal plates immediately after sorting
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 (two sequential gates):

  1. Gate 1 — SSC-A vs. FSC-A: Select cell events, discriminating bacteria from debris. Run a PBS-only control first to characterize the debris/non-cell scattering distribution, then set a stringent SSC-A threshold above the debris peak (~500 RFU) to reject contamination.
  2. Gate 2 — FSC-H vs. FSC-A: Select singlets (single cells), excluding doublets and aggregates.

Use single-cell mode (1 event per well) with purity sort mode to minimize contamination.

💡 Gating optimization: The primary cause of empty wells after sorting is debris contamination, not cell death. A debris peak at ~500 RFU SSC-A persists even through 0.22 µm filtration. More selective SSC-A gating eliminates this, boosting growth from 67% → 90.1%. Stringent gating is safe because sorted cell counts (~1,000–10,000) are far smaller than available cells (~107), so rare library members are not excluded.

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 ~70 wells/min (~500 events/sec)

4. Perform Sorting

  1. Load cell suspension into flow cytometer
  2. Set FSC/SSC gates to capture live bacterial population (see gating strategy above)
  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, centrifuge at 4,000 × g briefly to collect droplets at well bottoms
  7. Seal with adhesive foil seals (Thermo AB0626) and keep on ice between sorts
  8. Repeat steps 3–7 for additional plates

5. Outgrowth

  1. Incubate sorted plates at 37 °C for 17 hours, without shaking (important for observing single colonies at well bottom)
  2. Score growth by OD600 measurement: wells with OD600 > 0.05 are growth-positive
  3. Expect 67–90% of wells to show growth, depending on gating stringency (see gating optimization above)
  4. Prepare glycerol stocks during outgrowth: transfer 10 µL culture + 10 µL 50% glycerol to a fresh plate, seal, and store at −80 °C

Determining Sort Scale

The number of wells to sort depends on your library size and the synthesis skew of your oligo pool. More skewed libraries require higher oversampling to ensure full coverage of rare variants.

Use the interactive chart to estimate how many plates to sort for 90% library coverage. Adjust the library size and pool type to match your experiment, then see the recommended plate count.

For exact numbers, see usortm estimate in the CLI reference.

Library size:
Pool:

Quality Control

Troubleshooting

Problem Possible Cause Solution
Low growth rate (<67%) Debris contamination (primary cause), cell stress Optimize SSC-A gating threshold above the debris peak (~500 RFU); keep cells on ice; use fresher culture
Doublets or >95% growth Multiple cells per well Tighten FSC-H vs. FSC-A singlet gate, 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 a humidified incubator, avoid outer wells

Next Steps

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