Liver digestion protocol v2.0 — merge with AG Surace SOP #001

[Eomesodermin]

Merges the Abdullah lab liver protocol with AG Surace SOP #1 (S. Ng), keeping the gentleMACS route and adopting her filtration, Percoll and lysis steps.

New workflow: perfuse (ice-cold PBS) → gentleMACS → 70 um syringe-plunger filtration → wash spins → Percoll → ACK lysis.

MUS-LIV-DIG v1.2 → v2.0

Step	Change
STEP 2	Perfusion switched from MACS Buffer to ice-cold PBS, via IVC or portal vein. Callout: MACS contains 2 mM EDTA, which chelates the Ca2+/Zn2+ cofactors collagenase depends on and inhibits digestion. MACS remains correct for every subsequent step.
STEP 4	gentleMACS route now hands straight to filtration — no C-tube rinse into a tube. Manual dissociation retained and relabelled as a fallback.
STEP 5	70 um strainer + 5 mL syringe plunger directly into a 50 mL tube is now mandatory (previously only in the manual route, via a petri dish with dab/swirl recovery). Two 40 mL washes at 400 g replace the single wash.
STEP 5	50 g hepatocyte spin demoted to optional but recommended, with rationale: hepatocytes rupture at higher speeds, releasing DNA that drives clumping and DAMPs (ATP, HMGB1, mtDNA) that can activate the leukocytes being isolated. Trade-off against leukocyte loss stated.
STEP 5	All 400 g wash spins in 50 mL tubes 5 → 7 min; 15 mL tubes remain 5 min. Callout at first instance.
STEP 6	Percoll volume 10 mL in a 15 mL tube → 25 mL in a 50 mL tube. Spin 800 g / 20 min / acc 7 → 611 g / 15 min / acc 9. Warning that a 15 mL tube requires vacuum aspiration rather than pouring, or hepatocyte carryover is unacceptable. Post-Percoll wash removed — straight to lysis.
STEP 7	ACK standardised to 1 mL for 2 min, resolving a contradiction between the critical notes (2–5 min) and STEP 7 (30–60 s).
STEP 8	Final resuspension fixed at 5 mL; 96-well seeding guidance added.

Troubleshooting gains four rows covering hepatocyte carryover, EDTA-inhibited digestion, and the 50 mL spin-time trap.

Buffers

BUF-LIV-DIG (new) merges BUF-LIV-DIG-COL-D and BUF-LIV-DIG-COL-IV, which move to Archived_protocols/. Collagenase D 1 mg/mL, Collagenase IV 0.5 mg/mL, DNase I raised to 50 ug/mL for both (was 10 and 25). Base may be DMEM, PBS or HBSS.

BUF-PERC-37 v1.2 → v2.0. Density unchanged. Adds an explicit dual-convention section: 37% of isotonic stock and 33% final Percoll are the same solution, because SIP (Stock Isotonic Percoll) is 90% Percoll by volume. The AG Surace "33% Percoll" is the same gradient — its 9 mL Percoll + 1 mL 10x DPBS + 17 mL 1x DPBS is arithmetically identical. Callout warns that relabelling 37% as 33% without changing the recipe would drop the gradient to 29.7% final. Volumes updated to 25 mL per 50 mL tube with a clean per-liver scaling table (9.25 mL stock + 15.75 mL PBS per liver) and a tip to add margin for multi-liver preps.

Registry and dependency CSVs updated; MUS-LIV-DIG now depends on BUF-FACS-MACS, BUF-LIV-DIG, BUF-PERC-37, BUF-ACK-LYSIS.

Verification

Every recipe and scaling table is generated by an R chunk carrying stopifnot() assertions that the computed volumes reproduce the target concentrations and that components sum to the total — wrong arithmetic fails the render rather than printing a wrong number. The rpm↔RCF conversion (611 g = 1700 rpm, rmax ~18.9 cm) was validated against the published Eppendorf 5810R specification before use, and the protocol quotes RCF with a per-rotor rpm table so it transfers between centrifuges. All modified files render under Quarto; MUS-IEL-ISO, which also references BUF-PERC-37, still renders.

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