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Protocol

Complement-Dependent Cytotoxicity Assay

Thomas D. Duensing and Susan R. Watson

A useful feature of therapeutic antibodies is the ability to kill the cells to which they bind. Antibodies
are capable of mediating cell killing in a variety of ways. Apoptosis, complement-mediated mecha-
nisms, and antibody-dependent cellular cytotoxicity are all effects that can be assayed to characterize
lead antibody candidates. Extensive, multidose characterizations of a series of candidates can be
performed in a short amount of time using assays developed for high-throughput flow cytometry
systems. Antibodies that contain the Fc portion of the human IgG1 can activate complement-mediated
cell death. One way in which they do this is via direct complement killing of tumor cells by the
membrane attack complex, a process usually called complement-dependent cytotoxicity.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental
Health and Safety Office for proper handling of equipment and hazardous material used in this protocol.

RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at
http://cshprotocols.cshlp.org/site/recipes.

Reagents
Antibodies capable of binding to the target antigen of interest
Antibodies, negative control
Cell line expressing the target antigen of interest
Fetal bovine serum
Human AB serum (as a source of complement) (Sigma-Aldrich)
Opti-MEM cell culture medium, without pyruvate (ThermoFisher Scientific)
Phosphate-buffered saline (PBS) <R>
Propidium iodide, 1 µg/mL
Versene (ThermoFisher Scientific) (optional; see Step 1)

Equipment
Cell sieves, ~70-µm mesh
Flow cytometer with 96-well plate reading capability
Considerations for the selection of an appropriate flow cytometry system for a given application are discussed in
Introduction: Antibody Screening Using High-Throughput Flow Cytometry (Duensing and Watson 2018a).

Incubator, 37˚C
Plates, 96-well, U-bottomed, tissue culture-treated

From the Antibodies collection, edited by Edward A. Greenfield.

© 2018 Cold Spring Harbor Laboratory Press
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135
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T.D. Duensing and S.R. Watson

METHOD

Cell Preparation
1. If target cells are adherent, detach them from the culture vessel using Versene. Wash the cells once
with PBS by centrifugation at 1500 rpm for 3 min. Resuspend the cells in Opti-MEM
without pyruvate.
2. Filter cells through a 70-µm filter to remove aggregated cells. Count cells and adjust the concen-
tration to 1 × 106 cells/mL.
3. Seed 50 µL of cells into a clear U-bottom, 96-well tissue culture–treated plate.

Killing Assay
4. Add 50 µL of complement source (Human AB serum, diluted 1:4) and 50 µL of test antibody at
various concentrations to each well.
Typical test concentrations are 10, 2, 0.4, 0.08, 0.016, and 0.0032 µg/mL.

5. Incubate the cells for 2–3 h at 37˚C.

1000 1000
1.26 Dead
Dead cells 0.81
800 cells 800
32.2
47.3
600 600
53.5
SSC

SSC

400 400

200 200
Live cells
Live cells 6.47
0 0
0 200 400 600 800 1000 0 200 400 600 800 1000
FSC FSC

100

80
% Maximum

60

40

Dead cells
20

0
100 101 102 103 104
Propidium iodide

FIGURE 1. Sample gating strategy for determining complement-mediated cell killing. (Top left) Daudi cells cultured
with 10 µg/mL anti-KLH (negative control antibody) and human AB serum. Data are plotted as FSC versus SSC. (Top
right) Daudi cells cultured with 10 µg/mL rituxamab (positive control antibody) and human AB serum. Data are plotted
as FSC versus SSC. There is a 90% reduction in the cells in the live gate. The two scatterplots reveal that killing can be
detected without a dye. However, the addition of a viability dye such as PI makes the data easier to interpret. (Bottom)
Histogram showing an overlay of the PI staining of Daudi cells treated with anti-KLH and human AB serum (red tinted)
and rituxamab and human AB serum (blue line). These data are ungated.

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CDC Assay

6. Add 5 µL of propidium iodide.

7. Immediately analyze the cells using flow cytometry.

Analysis
8. Analyze the data by plotting forward scatter (FSC) versus side scatter (SSC), as well as a histogram
of the channel in which PI was detected (Fig. 1).
9. Plot antibody concentration versus percent dead cells (i.e., PI-positive cells) to determine the
EC50 of the tested antibody.

RELATED INFORMATION

Antibodies that contain the Fc portion of the human IgG1 can activate complement-mediated cell
death in one of two ways. The first, CDC, is described above. The second is antibody-dependent
cellular toxicity (ADCC) (see Protocol: Assessment of Antibody-Dependent Cellular Cytotoxicity by
Flow Cytometry [Duensing and Watson 2018b]).

RECIPE

Phosphate-Buffered Saline (PBS)

Final Final
Amount to add concentration Amount to add concentration
Reagent (for 1× solution) (1×) (for 10× stock) (10×)
NaCl 8g 137 mM 80 g 1.37 M
KCl 0.2 g 2.7 mM 2g 27 mM
Na2HPO4 1.44 g 10 mM 14.4 g 100 mM
KH2PO4 0.24 g 1.8 mM 2.4 g 18 mM
If necessary, PBS may be supplemented with the following:
CaCl2•2H2O 0.133 g 1 mM 1.33 g 10 mM
MgCl2•6H2O 0.10 g 0.5 mM 1.0 g 5 mM
PBS can be made as a 1× solution or as a 10× stock. To prepare 1 L of either 1× or 10× PBS, dissolve the
reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then
add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at
15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.

REFERENCES

Duensing TD, Watson SR. 2018a. Antibody screening using high-through- Duensing TD, Watson SR. 2018b. Assessment of antibody-dependent cellu-
put flow cytometry. Cold Spring Harb Protoc doi: 10.1101/pdb lar cytotoxicity by flow cytometry. Cold Spring Harb Protoc doi:
.top093773. 10.1101/pdb.prot093815.

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 Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on February 5, 2018 - Published by
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Complement-Dependent Cytotoxicity Assay

Thomas D. Duensing and Susan R. Watson

Cold Spring Harb Protoc; doi: 10.1101/pdb.prot093799

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