NLC Adoptive Transfer (Liver-derived NK-like Cells)

Dillon Corvino

Adoptive transfer of liver-derived NLCs for downstream phenotyping and functional studies, including planning calculations for donor requirements and standard timelines for LCMV- and BDL+LCMV-based experiments.
Published

January 9, 2026

Protocol ID: MUS-NLC-ACT
Version: v1.1
Author: Dillon Corvino

Purpose

This protocol describes adoptive transfer of liver-derived NLCs (NK-like cells) into recipient mice, with emphasis on:

  • Expected donor yield per liver and planning calculations for donor requirements
  • Target NLC transfer dose per recipient
  • Standard experimental timelines for:
    • LCMV-based experiments with prior P14 transfer
    • BDL + LCMV-WE experiments

Critical notes (read before starting)

  • NLC recovery varies by infection model, timepoint, operator, and digestion quality; plan donors with buffer.
  • Keep cells cold after final wash and minimize time between final resuspension and injection.
  • Use congenic markers (CD45.1/CD45.2) where possible to track transferred NLCs.
  • Final injection buffer should be sterile PBS unless internal SOP specifies otherwise.
  • All procedures must comply with approved animal licences and biosafety regulations.

Approximate timing

  • Donor liver harvest to single-cell suspension: ~2–2.5 h (see liver digestion protocol)
  • Enrichment / sort (if used): ~30–180 min (method-dependent)
  • Counting + preparation for injection: 10–20 min
  • Transfer (tail vein i.v.): ~1–2 min per mouse

Procedure

Standard yields and transfer dose (for planning)

Expected donor yield

  • Expected NLC yield per donor liver: 5,000–8,000 NLCs per mouse liver

Target recipient dose

  • Adoptive transfer dose: 60,000–80,000 NLCs per recipient

Donor requirement planning table

Assumptions for planning: - Low yield = 5,000 NLCs per donor liver - High yield = 8,000 NLCs per donor liver - Low transfer dose = 60,000 NLCs per recipient - High transfer dose = 80,000 NLCs per recipient - Donor livers required are rounded up to whole livers

Recipient transfer dose Donor yield per liver Donor livers needed per recipient Example calculation
60,000 8,000 8 60,000 / 8,000 = 7.5 → 8
60,000 5,000 12 60,000 / 5,000 = 12 → 12
80,000 8,000 10 80,000 / 8,000 = 10 → 10
80,000 5,000 16 80,000 / 5,000 = 16 → 16

Practical note: For multi-recipient experiments, multiply the “donor livers per recipient” by the number of recipients, then add an additional buffer (recommended) to account for losses during processing and sorting.

Donor cell preparation (high-level)

  1. Harvest donor livers from mice at the defined experimental timepoint.
  2. Prepare single-cell suspensions using the lab-standard liver digestion workflow (perfusion, enzymatic digestion, density enrichment, RBC lysis as needed).
  3. Enrich or sort NLCs according to the experimental design (bulk enrichment vs FACS for high purity).
  4. Count viable cells and prepare the final injection suspension in sterile PBS.
  5. Transfer 60,000–80,000 NLCs per recipient by i.v. tail vein injection.

Intravenous adoptive transfer parameters

  • Route: i.v. (tail vein)
  • Injection volume: 200 µL per mouse
  • Needle: insulin syringe, typically 27G–30G
  • Keep cells on ice and gently mix to maintain a uniform suspension during injection series.

Buffers Required

Full composition, preparation, storage, and safety information for each buffer lives in its own linked buffer document — only a concise per-buffer summary is reproduced here, generated automatically from that document.

None

Experiment timelines

LCMV experiment timeline (P14 → LCMV → NLC transfer)

Design: - P14 transfer on Day 0 - LCMV infection on Day 1 - NLC transfer 20 days after infection - Assessment 5 days after NLC transfer

Timeline table (absolute and relative)

Absolute day Relative description Event
Day 0 Start Transfer P14 cells
Day 1 24 hours later Infect with LCMV
Day 21 20 days after infection Transfer NLCs
Day 26 5 days after NLC transfer Harvest / assessment (organs, flow, etc.)

BDL + LCMV-WE infection timeline (BDL → LCMV → NLC transfer → harvest)

Design: - BDL on Day 0 - Infect with LCMV-WE approximately 9 days later - Transfer NLCs at 4–6 days post infection (dpi) - Harvest organs at 9 dpi

Timeline table (absolute and relative)

Absolute day Relative description Event
Day 0 Start BDL surgery
Day 9 ~9 days later Infect with LCMV-WE
Day 13–15 4–6 dpi Transfer NLCs
Day 18 9 dpi Harvest organs for analysis

Interpretation of spacing: - If NLC transfer occurs at 4 dpi, harvest at 9 dpi is 5 days after transfer. - If NLC transfer occurs at 6 dpi, harvest at 9 dpi is 3 days after transfer. - Choose dpi for transfer based on the desired window between transfer and analysis.

Typical readouts (minimal set)

  • Tracking of transferred NLCs by congenic markers (frequency, phenotype) in liver ± spleen/blood
  • Recipient immune context (key lymphoid/myeloid compartments as relevant to model)
  • Functional assays as needed (e.g., ex vivo stimulation / cytokines, cytotoxicity surrogates)
  • Infection-context readouts (use lab-standard viral load or surrogate readouts where applicable)

Safety (brief)

  • All animal procedures must comply with approved animal licences and institutional regulations.
  • Wear appropriate PPE (lab coat, gloves, eye protection) when handling animals, tissues, and reagents.
  • Handle sharps (needles, scalpels) with care and dispose of immediately in approved sharps containers.
  • Perform tissue and cell handling in a biological safety cabinet where appropriate.
  • Dispose of viral waste and contaminated materials according to local biosafety regulations.

Version history

Version Date Author Change summary
v1.0 2026-01-09 Dillon Corvino Initial NLC adoptive transfer protocol (planning yields, donor requirements, and standard timelines for LCMV and BDL+LCMV designs)
v1.1 2026-09-18 Dillon Corvino Replaced # Buffers used with an auto-generated # Buffers Required section (one heading + one small table per buffer, sourced from each buffer’s own summary: data), moved to the correct position after Procedure, and fixed leaked R package-startup warnings.