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Simple Staining Technique01:24

Simple Staining Technique

OverviewStaining techniques in microscopy enhance the visualization of microorganisms by increasing contrast and allowing the differentiation of cellular structures. Simple staining is one of the fundamental methods used to observe the basic morphological characteristics of microorganisms, including their size, shape, and arrangement. This method relies on the application of a single dye to stain the entire cell, producing a clear contrast between the cell and the background.FixationFixation is...

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Easy and Accessible Workflow for Label-Free Single-Cell Proteomics.

Ximena Sanchez-Avila1, Thy Truong1, Xiaofeng Xie1

  • 1Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States.

Journal of the American Society for Mass Spectrometry
|August 18, 2023
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A new, low-cost HP D100 Single Cell Dispenser enables accessible single-cell proteomics (SCP). This commercial platform offers accurate cell dispensing and rapid sample preparation, broadening SCP accessibility for researchers.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Molecular Biology

Background:

  • Single-cell proteomics (SCP) offers unique biological insights but is often limited by high costs and specialized equipment.
  • Current commercial platforms for SCP are expensive and require significant technical expertise, hindering widespread adoption.

Purpose of the Study:

  • To evaluate the HP D100 Single Cell Dispenser as a low-cost, accessible tool for label-free single-cell proteomics (SCP).
  • To assess the accuracy, reproducibility, and proteome coverage achieved using the HP D100 for SCP sample preparation.

Main Methods:

  • Utilized the HP D100 Single Cell Dispenser for isolating and preparing single cells in 384-well PCR plates for label-free SCP.
  • Employed centrifugation steps post-cell and reagent dispensing to optimize single-cell recovery and sample integrity.
  • Analyzed single-cell samples using both home-packed and commercial nanoLC columns.

Main Results:

  • The HP D100 demonstrated high accuracy and reproducibility for dispensing volumes between 200 nL and 2 μL.
  • Approximately 97% of dispensed wells yielded proteome coverage consistent with single cells after optimized centrifugation.
  • A fully commercial workflow using the HP D100 and well plates provided rapid, easy-to-use SCP sample preparation with no loss in proteome coverage compared to nanowell methods.

Conclusions:

  • The HP D100 Single Cell Dispenser, combined with commercial well plates and one-step processing, offers a rapid, user-friendly, and cost-effective workflow for SCP.
  • This approach significantly enhances the accessibility of single-cell proteomics to a broader range of laboratories.
  • While commercial columns reduced protein identification, the overall commercial platform presents a viable alternative for SCP sample preparation and separation.