DUPA-FITC

Flow cytometry (FC) is a technique for high-speed, step-by-step quantitative analysis and sorting of single cells or other biological particles in a suspension by detecting labeled fluorescent signals.

Flow cytometric DNA-content cell-cycle staining measures the fluorescence intensity of DNA-bound fluorochromes in single cells or nuclei to estimate DNA content distributions, allowing assignment of populations to G0/G1, S, and G2/M phases by DNA histogram deconvolution. Propidium iodide (PI) intercalates into DNA, and PI fluorescence is proportional to cellular DNA content when staining is performed under conditions that make DNA accessible and minimize non-DNA signal. Cells with G2/M DNA content are expected to show approximately twice the fluorescence intensity of G0/G1 cells, while S-phase cells occupy intermediate fluorescence values. PI-based DNA-content analysis can also detect cells with fractional DNA content, often reported as sub-G1, when DNA fragmentation and extraction during staining reduce retained DNA signal in apoptotic cells. DAPI is an alternative DNA fluorochrome for univariate DNA-content analysis, while bivariate approaches combining DNA content with proliferation

Density gradient centrifugation enriches cells by buoyant density: cells sediment during centrifugation until they reach a medium layer or interface compatible with their density, allowing mononuclear cells, granulocytes, erythrocytes, and density-defined subpopulations to be recovered from separate bands or layers. Classic blood-cell applications include Ficoll/sodium-metrizoate or Ficoll-Hypaque enrichment of peripheral blood mononuclear cells, Percoll subfractionation of PBMC and T-cell populations, and Percoll-based neutrophil isolation from whole blood or leukocyte-enriched suspensions. The readout is the physical recovery of enriched cell bands, followed by cell counting, morphology, viability, and immunophenotyping to determine yield, purity, and suitability for downstream assays.

Phospho-flow cytometry detects intracellular phosphorylated signaling proteins in single cells using phospho-specific antibodies after rapid fixation and permeabilization; the fluorescence intensity reflects phosphorylation state and therefore kinase-pathway activation, inhibition, or drug response in defined cell subsets. Unlike Western blot, phospho-flow preserves single-cell resolution and can measure signaling heterogeneity in cancer cells, primary immune cells, dissociated mouse tumors, macrophages, organoid-derived cells, and drug-screening samples when validated antibodies and fixation/permeabilization conditions are used.