Assays

Flow cytometry/FACS

• uses antibodies

• Forward vs side scatter

  • Forward scatter (FSC) correlates with cell size, while side scatter (SSC) reflects internal complexity/granularity.Lecture-20-2.pdf+1​

Fluorescence histograms: Fluorescently labeled cells are measured one‑by‑one in a laser beam; histograms of fluorescence intensity show distributions of marker expression or DNA content across the population

PCR

PCR uses repeated cycles of DNA denaturation, primer annealing, and extension by a thermostable DNA polymerase to exponentially amplify a specific DNA segment

FISH/RNA-ISH

FISH uses fluorescent DNA probes to hybridize specific DNA sequences in chromosomes or nuclei, localizing genes, copy number, or chromosomal abnormalities.Lecture-20-2.pdf​

RNA‑ISH uses labeled RNA/DNA probes to detect specific mRNA transcripts in cells or tissues, mapping spatial gene expression.

FRAP

FRAP: Fluorescence recovery after photobleaching uses a high‑intensity laser to bleach fluorophores in a defined region, then monitors recovery as unbleached molecules move in; the kinetics report molecular diffusion and binding dynamics in membranes or cytoplasm

FRET

FRET: Fluorescence resonance energy transfer occurs when an excited donor fluorophore transfers energy non‑radiatively to an acceptor fluorophore within ~1–10 nm, requiring spectral overlap and proper orientation; FRET changes report close molecular interactions or conformational changes (e.g., biosensors, protein–protein interactions)

DNA gel electrophoresis

Separates DNA fragments by size in an agarose gel under an electric field; smaller fragments migrate farther toward the positive electrode

Western blot

• uses antibodies

Combines SDS‑PAGE with transfer of proteins to a membrane and detection with specific antibodies

CRISPR: dCas9-eGFP

Clustered Regularly Interspaced Short Palindromic Repeats

Guide RNA that directs Cas9/dCas9 to a specific genomic DNA sequence via base pairing for CRISPR editing or regulation

catalytically dead Cas9 (dCas9) binds target DNA without cutting and can be fused to repressors (CRISPRi), activators (CRISPRa), or fluorescent proteins for gene regulation and imaging

LDH assay

Lactate dehydrogenase released into the medium from cells with ruptured membranes (necrosis or late apoptosis) is quantified enzymatically as a measure of membrane integrity loss and cytotoxicity

DNA dyes

DNA content stains: PI, DAPI, and Hoechst intercalate or bind DNA and allow quantification of DNA content by microscopy or flow cytometry, distinguishing G0/G1 (2N), S (between 2N and 4N), and G2/M (4N)

Optogenetics

Optogenetics introduces light‑gated ion channels or pumps (opsins such as ChR2 or NpHR) via viral vectors or genetic constructs so that specific neurons or cells can be activated or silenced by light, enabling precise control of circuit activity and behavior

Annexin V assay

Annexin V assay: Phosphatidylserine (PS) flips from inner to outer leaflet early in apoptosis; fluorescent Annexin V binds exposed PS, and combined Annexin V plus PI staining distinguishes viable (double‑negative), early apoptotic (Annexin V+ PI−), and late apoptotic/necrotic (Annexin V+ PI+) cells by flow cytometry

ICC/IHC

• uses antibodies

ICC (immunocytochemistry) uses antibodies on cultured cells to detect proteins with preserved subcellular localization​

IHC (immunohistochemistry) uses antibodies on fixed tissue sections to detect proteins in the context of intact tissue

IF

• uses antibodies

Direct immunofluorescence labels the primary antibody with a fluorophore

Indirect immunofluorescence uses an unlabeled primary and fluorophore‑conjugated secondary antibodies for amplification.Lecture21-Midterm3_Review.pdf​

FUCCI

FUCCI system: FUCCI uses fluorescently tagged cell cycle regulators (e.g., Cdt1 and Geminin fusions) that are degraded in phase‑specific patterns, labeling nuclei with different colors in G1 vs S/G2/M for live imaging of cycle dynamics.