Fig. 1. Schematic representation of B-cell purification by negative selection. Spleens are removed from anesthetized mice and dispersed through a nylon mesh to generate a single-cell suspension. The splenocytes are washed (following removal of erythrocytes by osmotic shock) and incubated with anti-CD43 and anti-Mac-1 antibody-conjugated microbeads (Miltenyi Biotec). The bead-bound cells (positive fraction) are separated from unbound cells (negative fraction) using an AutoMacs magnetic cell sorter. The magnetized column retains the positive fraction while the negative fraction containing resting B cells is collected in the flow-through. The positive fraction is later eluted for examination.
A negative-selection procedure developed by Miltenyi and colleagues (7) was chosen for purification of splenic B cells (Fig. 1) . This method relies on the fact that most leukocytes express CD43; resting mature B cells are exceptions. Expression of CD43 has been demonstrated on immature B cells, plasma cells, and some mature B1 cells, in addition to granulocytes, monocytes, macrophages, platelets, natural killer (NK) cells, thymocytes, and peripheral CD8pos and most CD4pos T cells (8, 9, 10, 11, 12). The heterogeneous cell population isolated from spleen was incubated with paramagnetic microbeads coupled to an anti-CD43 monoclonal antibody. To improve the removal of non-B cells (specifically myeloid contaminants), anti-Mac-1 (CD11b) beads were also included during the negative-selection procedure. The beads with unwanted cells attached were then removed using an AutoMACS computer-controlled magnetic cell sorter (Miltenyi Biotec, Auburn, CA). Resting, mature B cells remain relatively unperturbed during this procedure, and cell activation associated with positive selection is avoided.
Fig. 2. Composition of the cell pools before and after sorting. The effectiveness of the negative selection was evaluated by four-color FACS analysis using antibodies to CD43, Mac-1, CD3e, Gr-1, and B220. Cells were examined by forward scatter (FSC) and side scatter (SSC) to identify the viable cell fraction (first row). Each plot represents a typical analysis from 20 experiments; the percentage of cells expressing Mac-1 (macrophages), CD3e (T cells), Gr-1 (granulocytes), or B220 (B cells) is shown in the appropriate quadrants. Positioning of the cross-hair gates was done on control plots derived from cells stained only for B220.
The enriched cell population was subjected to multiparameter fluorescence-activated cell sorting (FACS) analysis to estimate the percentage of cells expressing B220, a marker present on cells committed to the B lineage (Fig. 2) (13, 14, 15). Contaminating cell populations that remained after enrichment were examined by staining the leukocyte markers Mac-1, CD43, Gr-1, and CD3 (Fig. 2). The enriched cell fraction contained approximately 47 x 106 cells/spleen, of which 96% expressed B220 (B220pos) (Table 1). Only small numbers of CD43pos/Mac-1pos cells were present. Blockade of the Fc receptor on splenocytes prior to incubation with magnetic beads did not improve yields significantly (not shown). Further characterization of the isolated cells is described below (see Cell Characterization).
To evaluate the proliferative status of the isolated B cells, cellular DNA content was determined by FACS analysis after staining with propidium iodide (Fig. 3) . More than 95% of the cells resided within the G0/G1 phases of the cell cycle. Propidium iodide staining of a proliferating B lymphoma cell line, WEHI-231, is shown for comparison; only 38% of these cells were in G0/G1 phases of the cell cycle.
Fig. 3. Cell cycle distribution of isolated splenic B cells and a cultured, mouse B-cell lymphoma, WEHI-231. Freshly isolated splenic B cells and WEHI-231 cells were stained with 1 mg/ml of propidium iodide and examined by FACS. The distribution of cells throughout the cell cycle was estimated from their total DNA content (measured by relative fluorescence intensity). Representative histograms are shown.
The Laboratory for Development of Signaling Assays (Veterans Administration Medical Center, San Francisco) and the Cell Preparation and Analysis Laboratory (University of Texas Southwestern Medical Center, Dallas) both isolate and culture B cells for AfCS experiments. It is essential that we are able to replicate these procedures at these sites. This will ensure that others in the research community can reproduce and extend our observations. The data presented in Table 1 demonstrate that the cell populations isolated in our two laboratories appear very similar. However, results from the Cell Preparation and Analysis Laboratory demonstrate a consistent trend of obtaining a greater number of starting cells per spleen and consequently greater yields of B220pos cells. We cannot currently offer a clear explanation for this difference.
Table 1. Summary of primary B-cell preparations. Cells were counted with a hemocytometer under a phase-contrast microscope. Viability was determined by trypan blue exclusion. B220pos cells were quantified by FACS analysis. Data are mean ± SD. The numbers in parentheses are the number of experiments in which the information was quantified. Calculated values are the average of specified determinations. ND=Not determined.
|Cell Prep and Analysis Lab†||Assay Development Lab|
|Body Weight (g)||ND||25.8 + 2.2||(190)|
|Spleen Weight (mg)||ND||84.1 + 12.8||(192)|
|Spleen Weight/Body Weight (x103)||ND||3.3 + 0.5||(190)|
|Viable (%)||81.9 + 3.6||(19)||79.2 + 5.4||(19)|
|Cells/Spleen (x106)||112.0 + 14.0||(19)||98.7 + 16.0||(20)|
|B220pos (%)||56.1 + 4.5||(19)||62.2 + 5.9||(16)|
|B220pos Cells/Spleen (x106)||62.5 + 9.2||(19)||63.3 + 13.9||(16)|
|Post-Sort Flow-through (B-Cell)||(19)|
|Viable (%)||90.4 + 4.0||(19)||95.1 + 1.9||(18)|
|Cells/Spleen (x106)||53.7 + 8.2||(19)||41.4 + 6.6||(20)|
|B220pos (%)||96.2 + 1.6||(19)||96.3 + 2.7||(20)|
|B220pos Cells/Spleen (x106)||52.2 + 7.7||(19)||39.8 + 6.4||(20)|
|82.8 + 8.2||(19)||65.4 + 16.8||(18)|
|Viable (%)||87.7 + 5.5||(19)||87.7 + 4.5||(7)|
|Cells/Spleen (x106)||36.7 + 11.2||(19)||29.7 + 5.9||(8)|
|B220pos (%)||15.0 + 3.0||(19)||26.6 + 1.7||(17)|
|B220pos Cells/Spleen (x106)||5.5 + 2.0||(19)||7.5 + 2.3||(8)|
| *Yield = (number of B220pos
cells in the flow-through)/(number of B220pos cells in the total
† All preparations were from 16 mice each.