Culturing and Harvesting Lymphocyte Chromosomes

for Fragile Sites

 

I. Purpose

A. To establish the presence of fragile (fra) sites on chromosomes. Fragile sites are defined as non-staining gaps of variable width, usually involving both chromatids, but always occurring at the same chromosome loci and are inherited in a Mendelian fashion.

B. Fragile sites are best shown by culturing cells in medium with a folic acid antagonist such as 5-Fluoro-deoxy-uridine (FUDR) or in media with a DNA synthesis inhibitor such as excess thymidine.

C. This method is especially useful for establishing the presence of a fragile site at Xq27 in patients with X-linked mental retardation.

II. Culture Procedure:

A. Obtain 2-4 ml of unclotted whole blood aseptically by venipuncture using a collection tube with sodium heparin. Invert the tube several times to prevent clotting. Assign accession number to specimen according to procedure in Laboratory Procedures Manual.

B. Aseptic technique must be used when setting up cultures, preferably under a laminar flow hood. To maximize the accuracy of this test, cultures are set up in both folate-deficient medium and regular medium with a folic acid antagonist. Using tape labels, label three T-25 flasks with the patient number, type of culture (1 flask 0.1 mcM FUDR, 1 flask 0.2 mcM FUDR, 1 flask Excess Thymidine), culture identification letter, and day of harvest (cultures are harvested after 3 days).

Example:

Pipet 10 ml of complete RPMI-1640 media into each flask. Using a syringe add 0.2 ml of Phytohemagglutinin solution to all three flasks. For the folic acid antagonist cultures, add 0.1 ml of FUDR to the first flask and 0.2 ml of FUDR to the second flask. Using a sterile pipet add 0.5 - 0.7 ml of whole blood to each culture. Seal flasks tightly, mix gently, and incubate cultures at 37 C for 72 hours. Add 0.1 ml Thymidine working solution to Excess Thymidine culture after 48 hours. Cultures should be gently mixed 2-3 times per day during incubation.

III. Harvest Procedure:

A. 1 1/2 hour before harvest add 65 mcl of colcemid working solution to each culture flask and mix.

B. Put the 0.075 M KCl hypotonic solution in the incubator to pre-warm. Prepare fixative of methanol:glacial acetic acid (3:1). Usually 40 ml of fix will be used per patient.

C. Gently swirl the flasks. Pour the contents of each flask into a 15 ml screw-cap conical centrifuge tube; transfer the tape label making sure no label mistakes occur. Centrifuge the tubes for 6 minutes at 150 x g (900 rpm).

D. Aspirate and discard all but 0.5 ml of supernatant, (note that the white blood cells will form a layer on top of the red cells and will be lost if too much supernatant is removed). Resuspend the cell pellet by mixing by hand, do not use a mechanical vortexer as this may break cells, this is especially important in step F. Add 5 ml of pre-warmed hypotonic solution and mix. If necessary, use a pasteur pipet to bubble air through the suspension to completely break up the pellet. Incubate the tubes at 37 C for 15-30 minutes.

E. Centrifuge for 6 minutes at 150 x g.

F. Aspirate and discard all but 0.5 ml of supernatant. Mix by hand to break up the cell pellet. It is important to get the cell pellet completely resuspended, but cells are fragile and are easily broken.

G. Slowly add 4 ml of fixative (2 pasteur pipetfuls) letting it run down the side of the tube so that it layers on top of the cell suspension. Cap the tube. Mix rapidly by hand so that all the cell suspension is fixed evenly; if it is mixed too slowly, clumps may form. Let sit at room temperature for 15 - 20 minutes.

H. Centrifuge for 6 minutes at 150 x g.

I. Aspirate and discard all but 0.5 ml of supernatant. Resuspend cell pellet. Add 4 ml of fixative and mix. Let sit at room temperature 15 - 20 minutes.

J. Repeat steps H,I 1 or 2 times until the pellet is clean and white. Culture is now ready to make slides. For best results, slides should be made the same day as the harvest.

IV. Slide Preparation:

See Regular Procedure for Culturing Lymphocytes.

V. Solutions:

See Regular Procedure for Culturing Lymphocytes, plus:

FUDR (5-Floru-deoxy-uridine): 2.46 mcg/ml dH2O, sterile filter, store at 4 C. To make a 0.1 mcMolar soln add 0.1 ml to 10 ml of culture media.

Thymidine Working solution: 30 mg/ml Thymidine in RPMI-1640 media, store frozen.

VI. Reagents:

See Regular Prodedure for Culturing Lymphocytes, plus:

FuDr: Sigma cat # F0503
Thymidine: SIGMA cat# T-9250, 5 g