67
Affinity columns were prepared as follows: The coupled Sephadex
G-20 preparations were washed with 5% FCS in G-MEM and 8 ml of packed
volume was poured under 1 x g into 12 ml disposable syringes. Two and
one half billion cells in 2.5 ml of 5% FCS in G-MEM were loaded di'op-
wise (10 drops/min) followed by the slow dropwise addition of 5% FCS
in G-MEM. Elutions were monitored periodically until the effluent was
cell free. The nonadherent cells were washed three times with medium
prior to further use.
Glass Wool Fractionation
The method described by Trizio and Cudkowicz (110) was adapted for
use in glass wool and nylon wool column fractionations of alligator
peripheral blood lymphocytes. Glass wool (Corning Glass Works, Corning,
N.Y.) was pretreated by rinsing three times with pyrogen free 0.15 M
NaCl, boiled 1 hr in tripled-distilled water (three changes) and dried
by lyophilization. Twelve milliliter disposable syringes were packed
to the 8 ml mark with the pretreated glass wool and sterilized. Prior to
loading cells on the prepared column, 40 ml of prewarmed (32C) G-MEM
was passed through the column followed by 15 ml of 5% FCS in G-MEM. The
column was then incubated for 30 min at 32C in 5% C02~95% air. One hun
dred million cells in 2 ml of 5% FCS in G-MEM were loaded onto each
column and were washed into the column with 1 ml of 5% FCS in G-MEM.
Loaded columns were incubated in a vertical position at 32C for 1 hr
in 5% C02~95% air. Nonadherent cells were eluted very slowly (20 drops/
min) with 20 ml of 5% FCS in G-MEM (32 C). Fifteen milliliters of warm
5% FCS in G-MEM were then slowly flowed through as a "buffer" between the
nonadherent and the adherent fractions. Care was taken not to gener
ate a fluid head of pressure nor to jar the column during the slow