Serum Selection for Cytotoxicity Assays — ADCC, CDC, MTT, AlamarBlue, LDH & ELISpot
Serum is not a neutral background in cytotoxicity assays. It is the most biologically active variable in your system — containing complement proteins, immunoglobulins, growth factors, and lipid-binding proteins that interact directly with effector cells, target cells, fluorescent readout reagents, and test compounds. The wrong grade does not add variability. It changes the signal being measured.
2. Active complement in non-HI serum causes spontaneous lysis of complement-sensitive cell lines — inflates background cytotoxicity.
3. FBS and BSA bind fluorescent dyes (AlamarBlue, calcein AM) — serum concentration must be identical in ALL wells including controls.
Quick Reference — Serum by Assay Type
| Assay Type | Recommended Serum | Standard FBS? | Critical Interference |
|---|---|---|---|
| ADCC (NK cell-mediated) | FBS Ultra Low IgG <5 µg/mL | ✗ No | Bovine IgG competes with mAb for FcγRIII on NK cells |
| CDC — active complement | Human Serum AB OTC (native) | ✗ No | Bovine complement ≠ human complement specificity |
| CDC — negative control | Human Serum AB Heat Inactivated | ✗ No | HI destroys complement — required paired with native for valid CDC |
| MTT / MTS | FBS Low Endotoxin ≤5 EU/mL | ⚠ Only LE | Endotoxin activates TLR4 → non-specific growth inhibition |
| AlamarBlue / Resazurin | FBS Low Endotoxin ≤5 EU/mL | ⚠ Only LE | FBS/BSA quench fluorescence — identical serum % in ALL wells |
| LDH release | FBS Low Endotoxin, low haemoglobin | ⚠ Only low Hgb | Haemolysis releases endogenous LDH → elevated background |
| ELISpot (IFN-γ, Granzyme B) | FBS Very Low Endotoxin ≤1 EU/mL or Human Serum AB HI | ✗ No | Endotoxin activates monocytes → non-specific spot background |
| Hepatotoxicity (HepG2, HepaRG) | FBS Low Endotoxin ≤5 EU/mL | ⚠ Only LE | Endotoxin activates hepatoma innate immune responses |
| 3D Spheroid cytotoxicity | FBS Ultra Low IgG <5 µg/mL (2–5%) | ✗ No | Bovine IgG interferes with anti-tumour antibody readout on spheroids |
| Neutralisation assay | FBS Very Low Endotoxin ≤1 EU/mL | ✗ No | Complement in non-HI FBS can neutralise enveloped viruses |
ADCC — Why FBS Ultra Low IgG is Non-Negotiable
ADCC measures NK cell-mediated killing of antibody-opsonised target cells via FcγRIII (CD16). Standard FBS contains 200–800 µg/mL bovine IgG which binds directly to FcγRIII — competing with the therapeutic mAb and reducing measured cytotoxicity. Controls cannot correct for this — the interference occurs before the readout.
| Parameter | Recommendation | Note |
|---|---|---|
| Serum grade | FBS Ultra Low IgG <5 µg/mL | Use in effector cell expansion AND assay medium — consistent throughout |
| Concentration | 5–10% | Identical in all wells including controls |
| Effector cells | PBMC or purified NK cells | Expand in FBS Ultra Low IgG for ≥5 days before assay |
| Target cell wash | 2× wash before assay | Remove residual bovine IgG from target cell medium |
| Readout | LDH release, calcein AM, or flow (PI) | Spontaneous release control must use identical serum concentration |
| Typical E:T ratio | 10:1 to 50:1 | Optimise per cell line and mAb concentration |
CDC — Why Human Serum AB OTC is Required
CDC measures antibody-mediated activation of the complement cascade leading to MAC formation and target cell lysis. Complement is highly species-specific — bovine complement has different specificity, activation kinetics, and regulatory protein interactions with human cells. Human Serum AB OTC (native, not heat-inactivated) provides active human complement.
| Anwendung | Serum | Why |
|---|---|---|
| CDC assay — active complement | Human Serum AB OTC (native) — 10–25% | Active human complement. Type AB — no anti-A/B lysis of target cells. |
| CDC assay — complement-depleted control | Human Serum AB HI — same concentration | 56°C/30 min destroys complement. Paired native/HI from same donor pool required for valid comparison. |
Three Serum Interference Mechanisms Explained
| Mechanism | Affected Assays | Serum Component | Solution |
|---|---|---|---|
| Fc Receptor Competition Bovine IgG occupies FcγRI/II/III on effector cells before mAb is added |
ADCC, ADCP, 3D spheroid antibody assays | IgG (200–800 µg/mL in standard FBS) | FBS Ultra Low IgG <5 µg/mL |
| Complement Activation Active complement lyses complement-sensitive cells spontaneously — independent of antibody |
CDC, ELISpot with PBMC, any assay with lymphoblast or RBC target cells | Complement proteins C1q–C9 (in non-HI serum) | Heat inactivated serum (56°C/30 min) or Human Serum AB HI |
| Dye & Compound Binding Serum proteins bind fluorescent dyes and test compounds — reduces apparent signal |
AlamarBlue, calcein AM, MTT, any fluorescent viability assay | Albumin, IgG, lipid-binding proteins | Identical serum % in all wells. Serum-only blank subtraction. FBS, endotoxinarme |
Validated Protocols
Protocol 1 — ADCC Assay with FBS Ultra Low IgG
1. Expand NK cells or PBMC for 5–7 days in 5–10% FBS Ultra Low IgG <5 µg/mL in RPMI-1640.
2. Prepare target cells in 5% FBS Ultra Low IgG — wash 2× before assay to remove residual bovine IgG.
3. Opsonise target cells with therapeutic mAb at 1–10 µg/mL for 30 min at 37°C.
4. Co-culture effector + target at E:T ratio 10:1–50:1 in 5% FBS Ultra Low IgG for 4–6 hours at 37°C.
5. Read LDH release, calcein AM, or PI flow — serum concentration identical in spontaneous and maximum lysis controls.
% Specific lysis = [(test − spontaneous) / (maximum − spontaneous)] × 100
Protocol 2 — CDC Assay with Human Serum AB (native + HI pair)
1. Prepare target cells (e.g. Daudi CD20+) in serum-free medium — wash 2×.
2. Add therapeutic mAb at 1–10 µg/mL — incubate 30 min on ice.
3. Add Human Serum AB OTC native at 10–25% as complement source. Pair with Human Serum AB HI at identical concentration as complement-depleted control.
4. Incubate 37°C, 60 min.
5. Read PI exclusion by flow cytometry or LDH release.
Complement-specific lysis = CDC (native) − CDC (HI)
Protocol 3 — AlamarBlue with Consistent Serum Control
1. Seed 2,000–10,000 cells/well in 96-well plate in 5–10% FBS Low Endotoxin ≤5 EU/mL.
2. Add test compound in serial dilution in medium with identical serum concentration.
3. Include: vehicle control, maximum kill control (0.1% Triton X-100), serum-only blank (no cells) — ALL with identical serum %.
4. Add AlamarBlue reagent at 1/10 volume — incubate 2–4 hours at 37°C.
5. Read fluorescence (Ex 560 / Em 590 nm). Subtract serum-only blank from all wells before calculating % viability.
Frequently Asked Questions
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FBS Ultra Low IgG and Human Serum AB samples available. Lot-specific IgG and endotoxin data per lot. Batch reservation for extended assay campaigns.
Email: info@seamlessbio.de | +49 851 37932226
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