Cytogenetic analysis relies on clear, well-defined chromosome banding patterns to detect structural and numerical abnormalities. Among these, GTG-banding remains the gold standard in human karyotyping. This method combines controlled proteolytic digestion with a differential DNA-staining dye to reveal alternating light and dark bands along each chromosome, allowing precise identification and mapping. In this guide, we walk through the complete GTG-banding protocol for peripheral blood lymphocytes, explaining not just the how, but the why behind each step so even beginners can understand the science driving the technique.

In routine peripheral blood lymphocyte cultures for karyotyping, T cells are stimulated because the mitogen used, phytohemagglutinin (PHA), specifically binds to receptors on T lymphocytes, triggering them to leave the resting phase and enter mitosis. T cells are more abundant than B cells in blood, giving higher and more consistent metaphase yields. B cells do not respond to PHA and require other mitogens, such as pokeweed mitogen or lipopolysaccharide, so they are only cultured in special situations like immunodeficiency workups or EBV transformation studies.

Materials

• Heparinized peripheral blood (sodium heparin — prevents clotting without harming cell growth)
  
• RPMI-1640 medium + L-glutamine + antibiotics
  
• PHA-M (phytohemagglutinin — mitogen)
  
• Colchicine
  
• 0.075 M KCl hypotonic solution (fresh, warmed to 37 °C)
  
• 3:1 methanol:glacial acetic acid fixative (freshly mixed, ice-cold)
  
• Trypsin 0.025% (w/v)
  
• Phosphate buffer pH 6.8
  
• Giemsa stain (1:20–1:30 in pH 6.8 buffer)
  
• Clean, grease-free glass slides
  

Step-by-Step

1) Set up lymphocyte culture

Do: Add 350 µL of heparinized blood to 4.5 mL RPMI-1640 complete medium containing:

• L-glutamine
  
• PHA-M
  
• Antibiotics (penicillin/streptomycin)
  Mix gently and incubate.
  

Why:

• Heparin keeps the blood from clotting but doesn’t interfere with the ions needed for cell signaling (unlike EDTA, which chelates Mg²⁺ and Ca²⁺).
  
• RPMI-1640 provides a balanced salt, glucose, and amino acid environment that mimics blood plasma — this keeps lymphocytes alive and happy for days.
  
• L-glutamine is a crucial amino acid that rapidly dividing cells use for building DNA, RNA, and proteins.
  
• PHA-M is a plant lectin that binds to receptors on T-lymphocytes, tricking them into thinking they’ve been activated by an immune signal. This makes them leave the resting phase (G₀) and start dividing.
  
• Antibiotics protect against bacterial contamination during the long 72-hour incubation.
  

2) Incubate for 72 hours at 37 °C, 5% CO₂

Why:

• 37 °C is human body temperature, the environment these cells are adapted to.
  
• 5% CO₂ keeps the pH stable in bicarbonate-buffered RPMI — CO₂ dissolves into the medium, forming a carbonic acid/bicarbonate buffer that holds pH at ~7.2–7.4.
  
• The 72-hour window gives enough time for many cells to reach metaphase without too many completing division and leaving the stage you need.
  

3) Add colchicine/Colcemid (metaphase arrest)

Do: Add enough stock to reach a final concentration of 0.05–0.1 µg/mL in your culture volume. Incubate for 30 min at 37 °C.

Why:

• Colchicine binds to tubulin, the protein that makes the mitotic spindle. Without a spindle, chromosomes can’t separate, so cells freeze in metaphase, the point at which chromosomes are most condensed and visible.
  
• The 30-min window traps enough cells in metaphase without over-condensing chromosomes, which can make banding less distinct.
  

4) First centrifuge (pellet the cells)

Do: Spin at 1000 rpm for 8–10 min. Discard the supernatant.

Why:

• Centrifugation collects the cells at the bottom of the tube (pellet) so you can change solutions.
  
• Gentle force is important, too low and you lose cells, too high and the pellet gets compacted and hard to resuspend, causing clumps later.
  

5) Hypotonic treatment

Do: Add 5 mL of freshly prepared 0.075 M KCl (pre-warmed to 37 °C) dropwise while gently mixing. Incubate for 20–30 min at 37 °C.

Why:

• Hypotonic means the salt concentration outside the cell is lower than inside. Water rushes into the cells to equalize concentration, swelling them and loosening the nuclear envelope. This lets chromosomes spread out instead of clumping.
  
• Fresh KCl avoids pH drift and contamination, ensuring osmotic pressure is exactly right.
  
• 37 °C makes swelling uniform as cold KCl slows water movement and causes uneven swelling.
  

6) Second centrifuge

Do: Spin again at 1000 rpm for 8–10 min. Discard the supernatant.

Why:

• Stops the hypotonic swelling at just the right point so cells are enlarged but not burst.
  

7) Fixation — first addition

Do: Add ice-cold 3:1 methanol:acetic acid dropwise to the pellet while gently mixing. Leave for 5–10 min.

Why:

• Methanol rapidly precipitates proteins and dehydrates the cell. This preserves the morphology of the structures.
• Acetic acid clears the cytoplasm and slightly swells the nucleus, making chromosomes more visible and spread out.
  
• Dropwise addition avoids osmotic shock that can cause cell clumping.
  

8) Fixation — 2 more changes

Do: Spin down, discard fixative, and replace with fresh cold fixative. Repeat for a total of three changes.

Why:

• Multiple changes wash out all traces of KCl and cell debris, preventing crystals and dirty backgrounds.
  
• Each change further hardens the chromatin, producing sharper bands later.
  

9) Store pellet

Do: Leave in 0.5–1 mL cold fixative at 4 °C until slide prep

Why:

• Cold storage stops any enzymatic activity and preserves morphology.
  
• A short “maturation” period in fix often improves chromosome spreading.
  

10) Slide dropping & air drying

Do: Bring suspension to room temp, resuspend gently, and drop from 20–30 cm onto clean slides. Let air-dry at ~50–60% humidity.

Why:

• Drop height controls spread: higher = more impact = better dispersion, but too high can damage chromosomes.
  
• Humidity affects drying speed: too dry = tight clumps; too humid = fuzzy chromosomes.
  

11) Trypsinization

Do: Dip slide in 0.025% trypsin for 10–30 s (time varies), then rinse in pH 6.8 phosphate buffer.

Why:

• Trypsin partially digests proteins holding DNA in place (histones and non-histone proteins). AT-rich DNA regions lose more protein and bind more dye → dark bands; GC-rich regions retain more protein → light bands.
  
• pH 6.8 rinse stops trypsin activity and restores optimal dye-binding conditions.
  

12) Giemsa staining

Do: Stain in 1:20–1:30 Giemsa in pH 6.8 buffer for 3–5 min. Rinse gently in buffer, quick dip in distilled water, air-dry, and coverslip.

Why:

• Giemsa contains azure B, which binds to the narrow minor groove of AT-rich DNA. Trypsin pre-treatment ensures differential accessibility, creating the G-banding pattern.
  
• pH 6.8 optimizes the electrostatic interaction between dye and DNA.
  

When performed correctly, GTG-banding produces sharp, reproducible banding patterns that form the backbone of clinical cytogenetics. Every step plays a critical role in ensuring quality metaphase spreads and reliable karyotypes. Understanding the reasoning behind each stage empowers cytogeneticists, especially those new to the field, to troubleshoot effectively and adapt the protocol to their laboratory conditions without compromising results.

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