The artical pertains to Functional Subclasses of t lymphocytes bearing different Ly Antigens. I do not understand what is being done to each cell? I know that antisera is a fluid phase of clotted blood that contains specific antibodies against the immunizing antigen but was is the immunizing antigen in this case?

Materials and Methods

Notations .The notation for congenic mice and antisera follows Shiku et al . (10) .
Mice .C57BL/6 (B6)’ (Ly phenotype 1 .2,2.2,3 .2) and B10.D2 were obtained from the Jackson
Laboratories, Bar Harbor, Maine, congenic lines B6/Ly-1 .1 (Ly-1 .1,2 .2,3 .2), B6/Ly-2 .1
(Ly-1 .2,2 .1,3 .2), and B6/Ly-2 .1,Ly-3 .1 (Ly-1 .2,2 .1,3 .1) from EAB (ref. 12, Table II) ; B10 .T(6R) and
AQR were bred by H. Cantor from breeders supplied by Dr . K. Melief of Tufts Medical School; (B6 x
BALB)F, (Ly-1 .2,2 .2,3 .2) was bred by H. Cantor .
Antisera .Anti -Ly-1 .2, anti-Ly-2 .2 and anti-Ly-3 .2, and anti-Thy-1 .2 are described in Shiku et al .
(ref. 10, Table III) . The Ly antisera we used, diluted 1 :10, were absorbed once with 120 x 10 8
syngeneic thymus + LNC/ml to remove autoantibody .
Complement (C)-Dependent Cytotoxicity Assay . 10-40 x 10 8 cells/ml ("Cr-labeled ; 30 min, 100
juCi/ml) were incubated with Ly antiserum diluted in phosphate-buffered saline with 5% fetal calf
serum (PBS-FCS) for 1/2 h at 37°C, washed once, brought up in 1 ml of freshly thawed rabbit serum
(diluted 1 :8 in PBS), and incubated for a further 1/2 h at 37°C . Rabbit sera selected for C were
preabsorbed with mouse cells in the presence of EDTA (see 10), which reduced background
cytotoxicity for spleen and LNC, under the conditions described above, to <12% .
Sequential Lysis with Different Ly Antisera and C .The proportions of cells displaying one or
more Ly antigens were estimated from the lytic effects resulting from sequential exposure to two
different Ly antisera ; the protocol using B6 LNC as target cells is illustrated (with controls) as
follows :

cpm (antiserum) – cpm (NMS)
x 100
cpm (freeze-thaw) – cpm (NMS) .
cpm (NMS)
$ cpm (freeze-thaw) x 100 (= 9% in both Steps 1 and 2 in this particular test) .
§ Cells from Step 1 were spun down and resuspended in fresh PBS-FCS before Step 2 .