Hemolytic Assay Protocol for C3

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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

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.

Hemolytic assay protocol for C3. (Creative Biolabs Original) Fig.1 Hemolytic assay protocol for C3.

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Published Data

The expression of PoC3 in Japanese flounder tissues with and without bacterial infection.Fig 2. PoC3 expression in Japanese flounder tissues in different bacterial infection situations 1

The article studies the immunological properties of C3 and its active fragment C3a (PoC3 and PoC3a) from a fish, Japanese flounder (Paralichthys olivaceus). PoC3 expression was found to occur in nine different tissues. PoC3 expression was upregulated upon bacterial challenge. In serum, PoC3 was found to be able to directly kill specific pathogens. When recombinant PoC3a was administered to Japanese flounder, an enhanced ability to protect against bacterial infection was observed. In vivo studies showed that the dissemination and colonization of E. tarda in Japanese flounder tissues was significantly reduced in the presence of rPoC3a, suggesting that rPoC3a activated the host immune response, thereby enhancing bacterial clearance. Together, these results show that PoC3 is crucial in complement activation and is necessary for teleost to defend against bacterial infection.

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Reference

  1. Wu, Meng, Bei-bei Jia, and Mo-fei Li. "Complement C3 and activated fragment C3a are involved in complement activation and anti-bacterial immunity." Frontiers in Immunology 13 (2022): 813173. Distributed under Open Access license CC BY 4.0, without modification.
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