Extracellular Matrix Extraction with the 96-Well Sonicator UIP400MTP

Traditional Extracellular Matrix (EM) extraction methods often involve multiple steps to disrupt the biofilm matrix. However, these techniques can be time-consuming and inconsistent, leading to variability in results. With the 96-well plate sonicator UIP400MTP, the EM extraction process is significantly facilitated, providing highly efficient, precise and uniform biofilm disruption in high-throughput formats. The UIP400MTP uses focused ultrasound to generate controlled cavitation, effectively breaking down the biofilm matrix while preserving the viability and integrity of biofilm-embedded cells. Using the UIP400MTP enhances the reproducibility and accuracy of downstream assays, such as metabolomic and proteomic analyses, viability tests, and antimicrobial susceptibility studies. By streamlining EM extraction, the UIP400MTP enables researchers to achieve consistent and high-quality results, offering deeper insights into biofilm dynamics and interactions with external agents.

Extracellular Matrix Extraction

The extracellular matrix (EM) is a critical component of biofilms formed by microorganisms such as Candida albicans. Composed of polysaccharides, proteins, lipids, and extracellular DNA, the EM provides structural integrity, mediates adhesion to surfaces, and contributes significantly to antimicrobial resistance. Extracting and analyzing the EM is essential for understanding biofilm biology, elucidating mechanisms of drug resistance, and identifying novel therapeutic targets.

Protocol for Extracellular Matrix (EM) Extraction from Candida albicans Biofilms Using UIP400MTP

This protocol focuses on extracting the extracellular matrix (EM) of static Candida albicans biofilms. Integrating the UIP400MTP sonicator to replace traditional scraping or enzyme-based steps for improved efficiency and reproducibility.

Materials Required

Step-by-Step Instructions for EM Extraction

1. Formation of Static Biofilm
   For static biofilms, inoculate C. albicans into the wells of a 96-well plate using RPMI-1640 medium. Each well should contain a consistent volume of inoculum (e.g., 200 µL per well).
   Incubate the plate at 37°C under static conditions for 24 hours to allow biofilm formation on the well surfaces.
2. Biofilm Preparation for Extraction
   Following incubation, gently aspirate and discard the spent medium from each well without disturbing the biofilm.
   Rinse each well carefully with sterile PBS (e.g., 200 µL) to remove loosely attached cells and planktonic debris. Repeat this step twice.
   Add fresh PBS or the extraction buffer (e.g., 200 µL) to each well to prepare for sonication.
3. Sonication with UIP400MTP
   Place the 96-well plate containing PBS or extraction buffer into the UIP400MTP sonicator tray. To place the multi-well plate correctly, follow the instructions of the manual.
   Sonicate for 5-6 minutes at a gentle setting (60% amplitude, pulse mode), ensuring uniform disruption of biofilm structure and release of EM components.
   Use temperature control of the UIP400MTP sonicator (temperature sensor and settings) to avoid excessive heating or sample damage.
4. Cation Exchange Resin (CER) Treatment
   Transfer the sonicated liquid from each well to a new 96-well plate.
   Add a twofold volume of CER suspension (prepared in PBS or buffer) to each well (e.g., 400 µL total volume per well).
   Seal the plate and incubate it on a plate shaker or rotator at 400 rpm for 3 hours to allow binding and isolation of EM components.
5. Separation and Filtration
   Centrifuge the plate at 2,000 × g for 10 minutes to separate the resin and cells from the supernatant.
   Carefully transfer the EM-containing supernatant from each well to a new 96-well plate.
   Filter the supernatant through a 0.22 µm filter using a plate-based vacuum manifold system or equivalent to obtain a clean EM extract.
6. Analysis of EM
   Use techniques such as UPLC-Q-TOF-MS for untargeted metabolite profiling, or employ specific assays (e.g., BCA for proteins, triglyceride, and carbohydrate quantification kits) to characterize EM components.

High-Throughput Extracellular Matrix Extraction

The high efficiency of extracellular matrix (EM) extraction with the UIP400MTP sonicator has revolutionized sample preparation for assays requiring accurate analysis of biofilm properties. The UIP400MTP uses ultrasonic waves to precisely and uniformly disrupt the biofilm matrix, enabling the effective release of EM components while preserving cell viability and integrity. This approach significantly enhances the reliability of assays such as biofilm viability tests, proteomic and metabolomic studies, and antimicrobial susceptibility evaluations.

For biofilm recovery assays, such as colony-forming unit (CFU) counting, EM extraction with the UIP400MTP ensures unobstructed access of reagents to biofilm-embedded cells. Similarly, proteomic and metabolomic analyses, such as UPLC-Q-TOF-MS, benefit from the high-yield isolation of proteins, lipids, and metabolites. The UIP400MTP also supports precise antimicrobial susceptibility testing, allowing for accurate determination of biofilm MIC and MBEC values. Furthermore, the efficient extraction facilitated by the UIP400MTP aids in enzyme activity assays, component quantification, and structural studies, offering valuable insights into the role of extracellular matrices in biofilm architecture, adhesion, and resistance mechanisms.

This high-throughput, reproducible extraction method optimizes EM preparation, ensuring superior results across a range of biofilm-related assays.