Principle Blood lymphocytes are stimulated by the mitogen phytohemagglutinin (PHA) to rapidly grow and divide such that three days  of culturing will yield late prophase or early metaphase cells of sufficient quantity and quality for clinical analysis. Colcemid  inhibits the formation of the spindle fibers. When added to the stimulated culture, it causes mitotically active cells to be  arrested in early metaphase. The synchronization of the cultures with methotrexate and thymidine allows large numbers of  cells to enter early metaphase near the time of the harvest. Addition of ethidium bromide can also produce chromosomes with  greater length and resolution by intercalating with the DNA, thus inhibiting chromosome condensation

Thymidine (Sigma #T‐5018) (see Safety warnings)

Working solution: Add 0.025g thymidine to 100mL Hanks’ (1×) for a final concentration of 10–3 M

 Filter sterilize with  a 0.2‐μm filtration unit

 Aliquot 20mL into a T‐12.5 blue‐capped flask. Store at 4 °C

Shelf life: 12 months

Use 0.1mL  (100μL)/5mL culture

 

Procedure

Blood setup and culture

Add 5mL Complete Culture Medium to each of three 15mL Corning polypropylene tubes. Label two of these tubes as

2A and one tube as 2AEB. Label with patient name, date, and laboratory identification information. An additional tube may be prepared and labeled as B culture depending upon study requirements. This culture will yield a better mitotic index but will not have as high‐resolution chromosomes

. At 4:00 to 4:30p.m., add heparinized whole blood to 5mL Complete Culture Medium using a 1mL pipette or Eppendorf pipette aid. Use 0.25–0.3mL for newborns, 0.3–0.35mL for individuals < 15 years old, and 0.35–0.40mL for individua >15years old. Cap tube and mix well

 Incubate cultures at 37°C for 48 to 96 hours. Incubation time is dependent upon the age of the patient, type of study and urgency of the results (48hours for fast results and all newborns and young children, 72 hours for best results for children and young adults, and 72–96 hours for adults and women who are pregnant)

 Add 0.05mL of 10–5 M methotrexate to cultures 2A and 2AEB at 4:00 to 4:30 p.m. the day before harvest. Incubate cultures at 37°C overnight. Culture 2A and 2AEB are methotrexate‐synchronized overnight and released for 5 hours

Culture B, if set up, is an unsynchronized culture, and is incubated until harvest time at 37 °C

 Add 0.1mL of 10–3 M thymidine to the 2A and 2AEB cultures at 8:30 a.m. the morning of the scheduled harvest. Mix well and re‐incubate at 37°C until harvest