Cell suspension preparation and handling.
Time of hypotonic treatment
For lymphocytes, 12-20 minutes incubation in 0.075 M KCl (hypotonic buffer) at room temperature or 37? C was usually sufficient. For fibroblasts, same amount of time using a 1:1 mixture of 0.4% KCl : 0.8% sodium citrate worked usually well. Hypotonic pretreatment induces swelling of the cells and bursts open the red blood cells from peripheral blood cultures allowing their separation. Longer hypotonic incubations (45-60 minutes) or the use of a more hypotonic buffer resulted in longer, thicker, stickier, less refringent chromosomes, very difficult to spread, yielding poor banding but still good FISH signals. Too short hypotonic treatment leaves cell membranes and cell/nuclear debris around the chromosomes, which do not spread properly and have a poor banding and hybridization quality.
High speed centrifugation
As cytogeneticists well know, during fixation of the cells there is a gradual decrease in size of the pellet with each centrifugation step. When the usual 15-50 ml tubes are centrifuged at 800-1000 rpm, after the supernatant/fixative is discarded, the inner walls of the tubes are always covered with a layer of cells. This indicates that not all cells are pelleted at this speed. To avoid loss of biologic material, centrifugation steps can be performed in 2 ml microfuge vials with rounded bottom, which are spinned at 6000 rpm for 2-5 minutes, in a tabletop microfuge. At the end of each centrifugation, a final burst of 14,000 rpm for 2-3 seconds further helps pelleting all cellular material. These high-speed centrifugation steps do not "damage" the cells or the chromosomes, which maintain their quality for both G-banding and FISH, and are especially useful when working with small, "precious" pellets. High-speed centrifugation can also be used in combination with the "classical" protocol. Cells from 2-10 ml peripheral blood culture, or trypsinized fibroblasts or tumor cells from a T-75 flask can be harvested in 15-50 ml tubes and centrifuged as usual at 1000 rpm. The pellet can be subjected to hypotonic treatment in the same tube, followed by the addition of a small amount of regular fixative. After the tubes are centrifuged as usual at 1000 rpm, the cell pellets can be transferred to small microfuge vials and all subsequent fixative washes can be done in these vials, with no further loss of cells.
This procedure was tested by dividing various pellets in two parts, one processed according to classical procedures and the other subjected to several high speed centrifugation steps. G-banding and FISH results using the two pellet halves were identical (not shown).
Long term storage of cell suspensions
Cell suspensions can be conveniently stored in 1.5 ml or 2.0 ml microfuge vials filled with fixative or ethanol, at -20? to -70? C for many years. FISH using cosmid probes worked well on fixative-stored pellets more than 10 years old. If cell pellets sit longer than one week in the freezer (-20? C), or one day at 4? C, it is recommended to change the fixative once, prior to slide preparation. If the pellets are stored and handled in 2 ml vials and are centrifuged in tabletop microfuges, the process of changing the fixative requires only 3-4 minutes.