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In This Article

  • Overview
  • Protocol
  • Disclosures
  • Materials

Overview

This video demonstrates label-free identification of lymphocyte morphology and biochemistry through 3D quantitative phase imaging. This technique allows for a thorough analysis of lymphocytes, uncovering their internal structures and properties without the need for labeling procedures.

Protocol

1. Flow Cytometry and Sorting of Lymphocyte Subtypes

NOTE: Sorting lymphocytes depending on cell type is essential for establishing the ground-truth (i.e., correct) cell type labels to train and test a cell type classifier in supervised learning. Flow cytometry, a gold standard method, is used to identify and separate lymphocytes.

  1. Make a mixture of surface marker staining antibodies in 100 µL of fresh RPMI-1640 medium [with 10% FBS, 0.1 µg of CD3e (17A2), CD8a (53-6.7), CD19 (1D3), CD45R (B220, RA3-6B2), and NK1.1 (PK136)] and 0.25 µg of CD4 (GK1.5) antibodies to target B, CD4+....

Disclosures

No conflicts of interest declared.

Materials

NameCompanyCatalog NumberComments
Flow cytometryBD BiosciencesAria II or III
Imaging chamberTomocube, Inc.TomoDish
HolotomographyTomocube, IncHT-1H
Holotomography imaging softwareTomocube, Inc.TomoStudio
Image-processing softwareMathWorksMatlab R2017b
Falcon conical centrifuge tubeThermoFisher Scientific14-959-53A15 mL
Phosphate-buffered salineSigma-Aldrich806544-500ML
Ammonium-chloride-potassium lysing bufferThermoFisher ScientificA1049201
RPMI-1640 mediumSigma-AldrichR8758
Fetal bovine serumThermoFisher Scientific10438018

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