In vitro cytology refers to the study of cells outside the body, typically in controlled lab environments such as petri dishes or culture flasks. This field is fundamental in biomedical sciences for understanding cell behavior, pathology, drug responses, and mechanisms of disease. It involves a wide variety of techniques for detection, quantification, imaging, and functional analysis of cellular and molecular components. These methods are pivotal for diagnostics, therapeutic evaluations, and research in cellular biology, oncology, immunology, and tissue engineering.

Tests in in vitro cytology typically fall into four major groups:

First Group of Tests: Qualitative – Detection/Identification

1. PCR – Detects presence of a gene (DNA-based).
2. RT-PCR – Amplifies cDNA from RNA to identify gene expression.
3. Nested PCR – Two-step PCR for increased specificity and sensitivity.
4. Multiplex PCR – Simultaneous detection of multiple genes in one reaction.
5. Digital PCR (qualitative) – Detects presence/absence of target DNA/RNA molecules by analyzing droplets.
6. Southern Blot – DNA detection via labeled probe on membrane.
7. Northern Blot – RNA detection using a labeled probe.
8. Western Blot (qualitative) – Protein detection using antibodies.
9. Dot Blot – Protein or nucleic acid detection using spots and antibodies/probes.
10. Immunohistochemistry (IHC) – Protein detection in tissues using antibodies and chromogenic stains.
11. Immunocytochemistry (ICC) – Protein detection in cells using labeled antibodies.
12. Immunofluorescence (qualitative) – Detection of molecules using fluorescent antibodies in cells/tissues.
13. Flow Cytometry (phenotyping) – Surface marker detection using fluorescent antibodies.
14. FISH (fluorescence in situ hybridization) – Chromosomal localization of DNA/RNA using fluorescent probes.
15. In situ hybridization (ISH) – Detection of nucleic acids in cells/tissues using labeled probes.
16. ELISA (qualitative) – Colorimetric enzyme-based detection of antigens/antibodies.
17. Lateral Flow Assay – Rapid detection (e.g., at-home tests) via immunochromatography.
18. ELISPOT – Spot-based detection and quantification of cytokine-secreting cells.
19. MALDI-TOF microbial ID – Identification of microbes via mass spectrometry.
20. Gram Staining – Differentiates Gram-positive and Gram-negative bacteria by staining their cell walls.

Second Group of Tests: Quantitative – Measurement

1. qPCR (RT-qPCR) – Quantifies cDNA (RNA expression) using fluorescence.
2. Digital Droplet PCR – Absolute quantification of DNA molecules via partitioning.
3. ELISA (quantitative) – Measures antigen concentration using color intensity and standard curve.
4. Bradford Assay – Protein concentration by dye-binding color change.
5. BCA Assay – Protein quantification using reduction-based purple color generation.
6. UV-Vis Spectrophotometry – Quantifies nucleic acids/proteins by absorbance.
7. HPLC – Separation and quantification of sample components.
8. LC-MS/MS – Mass spectrometry with chromatography for precise compound quantification.
9. RNA-seq – Quantitative gene expression analysis under different conditions.
10. Gene Expression Microarray – Measures mRNA levels across thousands of genes.
11. Protein Microarray – Quantifies multiple proteins using immobilized antibodies.
12. Spectrophotometric Enzyme Assay – Measures enzyme activity via absorbance change.
13. Caspase Activity Assay – Quantifies apoptosis via caspase activity.
14. Cell Counting (Hemocytometer/Counter) – Direct cell number measurement.
15. Flow Cytometry (MFI) – Measures expression levels based on fluorescence intensity.
16. Alamar Blue Assay – Metabolic activity of cells via color/fluorescence change.
17. Simoa (Digital ELISA) – Ultra-sensitive protein quantification using femtomolar detection.
18. Immunoturbidimetric Assay – Measures protein concentration by turbidity from antigen-antibody complexes.
19. Mass Spectrometry Targeted Quantification – Exact peptide/metabolite quantification using isotope standards.
20. NanoDrop (DNA/RNA quantification) – Measures purity and concentration via UV absorbance.

Third Group of Tests: Localization/Imaging

1. Immunofluorescence Microscopy – Visualizes proteins in cells/tissues using fluorescence.
2. Confocal Laser Scanning Microscopy – High-resolution optical sectioning of specimens.
3. Two-Photon Microscopy – Deep tissue imaging with reduced photodamage.
4. Super-Resolution Microscopy (e.g., STED) – Imaging beyond the diffraction limit.
5. PALM/STORM – Nanoscale imaging using single-molecule resolution.
6. TIRF Microscopy – Surface-near imaging using evanescent waves.
7. FLIM – Measures fluorescence lifetimes to infer molecular environment.
8. TEM (Transmission Electron Microscopy) – Ultra-structural imaging at nanometer resolution.
9. SEM (Scanning Electron Microscopy) – 3D imaging of surface topography.
10. AFM (Atomic Force Microscopy) – Topographic imaging at molecular scale.
11. Live-Cell Imaging – Tracks dynamic processes in living cells.
12. GFP-Fusion Imaging – Tracks proteins fused with GFP in live cells.
13. Immunohistochemistry Imaging – Visual localization of proteins in tissue sections.
14. FISH Imaging – Visualizes chromosomal DNA/RNA with fluorescent probes.
15. In Situ Hybridization Imaging – Detects RNA/DNA within cells or tissues.
16. Histochemical Staining (H&E, PAS) – General tissue structure staining.
17. Micro-CT – 3D imaging of small tissue structures using X-rays.
18. Small-Animal MRI – High-resolution soft tissue imaging in animal models.
19. PET Imaging – Tracks radiolabeled molecules in live organisms.
20. Bioluminescence Imaging – Measures light emission from luciferase-expressing cells in vivo.

Fourth Group of Tests: Functional Assays

1. MTT Assay – Measures cell viability via mitochondrial reduction.
2. XTT Assay – Soluble version of MTT for easier viability readout.
3. WST-1 Assay – Highly sensitive metabolic activity assay.
4. Alamar Blue Assay – Fluorescent metabolic viability test.
5. Annexin V/PI Apoptosis Assay – Differentiates apoptotic vs necrotic cells using staining.
6. TUNEL Assay – Detects DNA fragmentation during apoptosis.
7. BrdU Incorporation Assay – Measures DNA synthesis during cell proliferation.
8. EdU Assay – Faster, click-chemistry version of BrdU for proliferation analysis.
9. Colony Formation Assay – Assesses clonogenic survival in adherent cells.
10. Soft Agar Assay – Evaluates anchorage-independent tumor cell growth.
11. Scratch Wound Healing Assay – Measures cell migration across a wound gap.
12. Transwell Migration Assay – Quantifies migratory response across a membrane.
13. Matrigel Invasion Assay – Measures invasive potential through ECM-like gel.
14. Tube Formation Assay – Assesses angiogenic potential of endothelial cells.
15. Phagocytosis Assay – Measures uptake of particles by immune cells.
16. ROS Detection (DCFDA) – Detects reactive oxygen species as fluorescence.