Hemolytic Assay Protocol for C3

Through our years of expertise in complement system research, Creative Biolabs steadily builds specific platforms and reliable methodologies. We can give extensive and personalized service to our international clients in the field of complement systems.

Introduction of C3 and Hemolytic Assay

Complement component 3 (C3) is a protein contributing to the innate immunity of the immune system and is encoded by the C3 gene on human chromosome 19. Activated forms of C2 and C4 consist of the heterodimer C3-convertase, also known as C4b2a, which can proteolytically process C3 to generate mature proteins C3a and C3b. C3 is essential for the complement system's activation. A hemolytic assay is a useful option for assessing C3 activity in the complement system, which can be used to assess the potency and toxicity of various compounds and to quantify this capability by lysing red blood cells in response to the chemical.

Materials for Assay

• 96-well U-bottomed plate
• Human C3-depleted serum (C3D)
• Normal human serum (NHS)
• HEPES-buffered saline buffer
• 2% Antibody-Sensitized Sheep Erythrocytes (SRBCs)

Protocol of Hemolytic Assay for C3

1. Prepare a serial dilution of NHS in a 96-well U-bottom plate. Repeat this procedure for the serial dilution of C3D.
2. Prepare the 100% lysis controls (ddH₂O) and 0% lysis controls (HBS).
3. Add HBS in each well in step 1. Add ddH₂O or HBS to the 100% lysis controls and 0% lysis controls, respectively.
4. Add 2% SRBCs to each well.
5. Seal and incubate the plate at 37°C. To stop the reaction, centrifuge the plate at 4°C.
6. Transfer the supernatant from each well to a new plate. Do not disturb the pelleted SRBCs. Record the 405 nm absorbances (A₄₀₅) by a plate reader.
7. Verify controls to show 100% (Maximum) and 0% (Background) lysis, respectively.
8. Determine the % Lysis in samples and calculate the 50% hemolytic complement (CH50) activity.

Fig.1 Hemolytic assay protocol for C3.

Creative Biolabs has focused on complement systems for decades and summarized well-defined methodology in this field. We still have the advanced experimental equipment and the perfect experimental platforms to meet and tailor all your demands for complement system research. For details about your project involving complement systems, feel free to contact us.

Services and Products at Creative Biolabs

Complement Products

Complement Test Services

• Hemolytic Assay Protocol for C1 Complex
• Hemolytic Assay Protocol for C4
• Hemolytic Assay Protocol for C2
• Hemolytic Assay Protocol for Terminal Components