Methods and Protocols

All standard methods and protocols used for studies described within this report are listed. Detailed recipes for preparation of solutions are also available and specified within each protocol. Additional methods or modifications to standard protocols are described below the table.

Mice

Hemizygous bcl-2^+/TGN (Em-bcl-2-22) transgenic mice were originally obtained from JAX Mice (http:// jaxmice.jax.org, Jackson Laboratory, stock no. 002319) and were maintained by breeding with C57BL/6 (B6) mice at the vivarium at the San Francisco VA Medical Center. These mice are currently maintained by JAX Mice only as frozen embryos. Hemizygous offspring were identified by using a PCR specific for the C-terminus of the human bcl-2 gene.

Genotyping by PCR

Genomic DNA from mouse tail ends (~5 mm length) was prepared by using Qiagen DNA extraction kits (QIAamp DNA mini kit, cat. no. 51306). Extracted DNA was tested for the presence of the human bcl-2 transgene and as a positive control, mouse IL-2, by using the Jackson Laboratory protocol for TgN(BCL2)22WEHI (http:// jaxmice.jax.org, genotyping protocol for stock no. 002319) using Amplitaq Gold (Roche, cat. no. 1699121) and a Gene Amp PCR System 9600 thermal cycler (Perkin-Elmer). This procedure identified both the PCR products for hbcl-2 (170 bp) and for mouse IL-2 (315 bp). Hemizygotes made up 50% of litters, consistent with previous demonstration that the transgene is inserted at a single (unidentified) site in this line (5).

Isolation of B Cells

The AfCS protocol, Isolation of Resting B Lymphocytes from One or More Groups of Four Mouse Spleens (PP00000001), was used with the adjustment that one Bcl-2^+/TGN spleen was processed as the equivalent of two B6 spleens. We avoided the use of Fc receptor (FcR) blocking antibodies during magnetic bead labeling, because we did not want the B cells exposed to these reagents.

Staining Cells for FACS Analysis

The AfCS protocol, Characterization of Cells by Flow Cytometry (PP00000018), was used. For staining of intracellular proteins such as bcl-2, we followed the manufacturer's instructions (BD kit, cat. no. 556357). Briefly, cells were manipulated at 4^oC in PBS/2 mM EDTA/0.5% BSA. Fc receptor blocking and staining of cell surface receptors was done prior to fixing and permeabilization for staining of human or mouse bcl-2, and blocking agents were included during staining with fluorophore-coupled primary antibodies. The intensities of fluorescence staining signals obtained using antigen-specific antibodies directly coupled to fluorophores were always compared with signal intensities obtained using isotype-matched negative controls (e.g., antibodies raised against keyhole limpet hemocyanin [KLH]).

B-Cell Culture

Isolated B cells were cultured in either Supplemented Iscove's Modified Dulbecco's Medium (SIMDM, AfCS solution protocol ID PS00000056) or SIMDM with 10% FBS (Hyclone), as specified. Cells were cultured at a density of 6 x 10⁶/ml, plating 3 ml, 0.6 ml, or 0.1 ml in 6-well, 24-well, or 96-well Costar ultralow plates, respectively (Fisher Scientific cat. no. 07-200-601, 07-200-602, and 07-200-603). For the culture of cells for more than three days, half of the culture medium was replaced with fresh medium on days 3 and 6 of culture. Cells were not cultured for longer than nine days.

Ligands

AfCS ligands anti-IgM, SDF-1a, BLC, anti-CD40, and IL-4 were made up according to AfCS protocols (PL00000006, PL00000003, PL00000005, PL00000001, PL00000004). Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 was purchased from Sigma-Aldrich (cat. no. L-4391). A stock solution of LPS was made by dissolving 1 mg (contents of 1 vial) in 1X PBS-0.1% BSA (PS00000082) to give a 1 mg/ml solution, which was aliquoted and stored frozen at -20^oC.

Calcium Assay

Ninety-six-well plate calcium (Ca²⁺) flux assays were performed using the FLEXstation (Molecular Devices) with modifications of two AfCS protocols PP00000011 (for suspended B cells) that is used with the Fluoroskan Ascent), and PP00000210 (for adherent RAW264.7, that is used with the FLEXstation). The FLEXstation, when operating on flex mode for robotic additions of ligands from a compound plate, excites and reads the assay plate from the bottom. Thus, to convert fluorescence units into calculated [Ca²⁺]_i values requires an equation that takes into account cell numbers being analyzed (given in PP00000210). This is in contrast to the measurements taken for suspended B cells using the Fluoroskan Ascent operated for a top read/total well assessment. Hence, modifications were made to load and plate nonadherent B cells and use minimum (extracellular calcium depleted, PS00000607) and maximum (excess extracellular calcium added, PS00000608) readings obtained using the procedure designed for assessments on adherent RAW 264.7 cells (PP0000210) to allow calculation of [Ca²⁺]_i values, assuming a K_d of 390 nM for fluo-3. Briefly, cells were loaded with the Ca²⁺ indicator dye, fluo-3 (3mM, Molecular Probes), in the presence of pluronic F-127 in SIMDM medium. Cells were loaded for 30 minutes with fluo-3 in a polypropylene tube in the dark at room temperature and mixed during loading by inverting the tube every 10 minutes. After loading, cells were pelleted, resuspended in assay buffer (HBSS-BSA, PS00000032), plated into 96-well assay plates (1 x 10⁶ cells/well, in 75ml) for use in the FLEXstation, and then placed in a humidified incubator at 37^oC. Cells were left for 1 hour after plating to allow equilibration and settlement of cells to the bottom of the wells of the assay plate before Ca²⁺ flux assays were performed.

Phosphoproteins

Samples were prepared, run on SDS-PAGE gels, and Western blotted according to AfCS Protocol PP00000010. Western blots were probed with antibody mixes developed by the AfCS, PS00000312 and PS00000334, and with positive control samples, PS00000083 and PS00000085, respectively, for comparison.

Proliferation Assay

Freshly isolated splenic B cells (prepared using AfCS Protocol PP00000001) in MACS buffer (PBS/2 mM EDTA/0.5% BSA, PS00000001) or cultured B cells were harvested and resuspended in SIMDM (PS00000056) for assay at 6 x 10⁵ cells/well. Ligands were added at the desired concentration to give a final volume of 200 ml/well, then 10 ml (1 mCi) of [³H]-thymidine (Perkin-Elmer, cat. no.NET221X) was added per well, and assay plates were incubated overnight in a humidified atmosphere at 37^oC with 5% CO2. The next day, plates were either harvested immediately and counted using a 96-well plate harvester/b-counter system (MicroBeta Tomtec, Perkin-Elmer) or were frozen for harvesting at a later time.

Migration Assay

Transwell migration assays were performed using 5 mm transwell inserts (Costar) suspended by the outer rim within individual wells of 24-well ultralow plates (Costar), so that B cells did not stick to the plates after migration through the transwells into the lower well; this was a problem with some tissue culture-treated dishes. Use of the ultralow adhesion plates allowed all the migrating cells to be harvested for counting with minimal stress. The wells of the 24-well ultralow plates were filled with 0.6 ml of assay medium, SIMDM (PS00000056), with or without chemokine added. 1 x 10⁶ B cells were added in 100 ml to the top transwell, which was placed in contact with the medium in the wells of the 24-well plates. The migration assay plates were placed in a humidified incubator (37^oC, 5% CO2) for 4 hours. After 4 hours, the transwells were removed, medium and cells from the lower wells were harvested, and the wells were rinsed once with 0.5 ml MACS buffer (PS00000001). Harvested cells with rinses were pelleted by centrifugation; cells were then resuspended in a small volume of MACS buffer (50-100 ml) and left on ice until counted. The numbers of viable cells present per sample were determined by counting cells able to exclude trypan blue.

Cell Culture and RNA Isolation for DNA Microarray Experiments

For analyses of gene expression, B cells were cultured for varying periods of time in SIMDM (PS00000056) -/+ 10% FBS (Hyclone, cat. No. SH30070.03) in 6-well Costar ultralow plates (Fisher Scientific, cat. no 07-200-601) at 6 x 10⁶ cell/ml, 3 ml per well. For ligand stimulation, cells were harvested from the 6-well plates, counted for viability, and replated at 18 x 10⁶/well in 12-well Costar plates. Cells were rested for 1 hour prior to ligand stimulation with a final volume after ligand addition of 1.2 ml/well, cell density at 16.7 x 10⁶/ml. Cells were treated with ligands for specified times and then harvested for RNA isolation (PP00000009). RNA quality was determined by running samples out on agarose gels (PP00000025). Microarray experiments used transcript profiling analyses to examine relative gene expression in paired samples (PP00000019), as described in the text (see sections: Gene Expression Changes in Response to In Vitro Culture and Anti-IgM Stimulation, above, and DNA Microarray Data Analyses, below).

DNA Microarray Data Analyses

In the analysis of Agilent array microchips, which were probed with competing red and green labeled probes derived from the original RNA samples, features with a saturated, below background, or less than 300 pixels green or red intensity in a given array were removed. For a given hierarchical clustering analysis, only features that had at least 80% nonblank measurements and an average of twofold or more changes in at least one experimental condition were included. Hierarchical clustering was done with the Multiple Array Viewer developed by The Institute of Genetic Research (TIGR). Euclidian distance was used as the similarity metric. The statistical significance of coclustering of genes involved in the same biological function/process was computed with CLASSIFI analysis (http://pathcuric1.swmed.edu/pathdb/classifi.html).

Lentivirus Production and Transduction

We used a combination of three plasmids, including a vector plasmid, pFUGW (14), with the pCMVDR8.91 packaging plasmid (31, 34), and the pMD.G envelope plasmid (31, 34), to produce nonreplicating HIV-based lentivirus following transfection of 293T packaging cells. The vector plasmid contained viral RNA packaging signal sequence and GFP marker protein between self-inactivating viral long terminal repeats (SIN-LTRs). The three LTRs were altered by deletion to become SIN-LTRs that are self-inactivated upon reverse transcription and integration after infection of the target host cell. pFUGW came from the laboratory of David Baltimore, and in this plasmid eGFP is expressed under the ubiquitin C promoter between viral SIN-LTRs. The pCMVDR8.91 'packaging' plasmid (Trono lab, Geneva) expresses HIV gag, pol, and rev genes, but does not carry viral RNA packaging sequence, so these genes do not get carried in the nonreplicating lentivirus that is produced. The envelope plasmid, pMD.G (Trono lab, Geneva) expresses vesicular stomatitis virus (VSV) G envelope protein, which binds cell membrane lipids and, hence, permits entry of the pseudotyped lentivirus produced into all mammalian cells. 293T packaging cells were transfected with three lentiviral plasmid combinations using the Lipofectamine 2000 (Gibco-BRL, Life Technologies, Inc.) following AfCS protocol PP00000200. The lentivirus preparations produced were used with and without concentration (PP00000202), and infections of either stimulated or nonstimulated B cells and the Ba/F3 pro-B cell line were performed in the presence of polybrene (Sigma-Aldrich, cat. no. P1274 at 4 mg/ml), according to AfCS protocol PP00000215.