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" Spleens were obtained from three normal untreated BALB/cALIZ (N9F1) mice or five pristane-treated BALB/c.ALIZ (N9F1) mice 67 days after the first injection of pristane. Splenic cells from two additional groups of mice, three untreated mice and five pristane-treated mice, were used to fractionate B220+ B cells from B220" non-B cells, using MACS. b Number of mice.

c Mice were left untreated or injected with Pristane on days 1 and 60. d Number of blue plaques (plaque-forming units, PFU) with mutations in gene lacl. e Number of total plaques.

^Mean mutant frequency determined as the ratio of mutant to total plaques, as multiples of io-5.

s Standard deviation of the mean mutant frequency, as multiples of 10~5. h Significance values were determined with the two-tailed paired t test. Mutant rates in fractionated cells were compared with mutant rates in whole spleens (NS, not significant).

" Spleens were obtained from three normal untreated BALB/cALIZ (N9F1) mice or five pristane-treated BALB/c.ALIZ (N9F1) mice 67 days after the first injection of pristane. Splenic cells from two additional groups of mice, three untreated mice and five pristane-treated mice, were used to fractionate B220+ B cells from B220" non-B cells, using MACS. b Number of mice.

c Mice were left untreated or injected with Pristane on days 1 and 60. d Number of blue plaques (plaque-forming units, PFU) with mutations in gene lacl. e Number of total plaques.

^Mean mutant frequency determined as the ratio of mutant to total plaques, as multiples of io-5.

s Standard deviation of the mean mutant frequency, as multiples of 10~5. h Significance values were determined with the two-tailed paired t test. Mutant rates in fractionated cells were compared with mutant rates in whole spleens (NS, not significant).

involved in antioxidative defense. To minimize this problem, mice should be stressed as little as possible before sacrifice, which should be performed by cervical dislocation rather than asphyxiation in CO2. For the fractionation of mouse B cells, we prefer MACS over fluorescence-activated cell sorting (FACS), despite the somewhat lower purity obtainable by MACS. The main benefit of MACS is the speed and ease of the procedure. Among the various protocols for B cell purification by MACS, we favor the positive enrichment afforded by the B220 (CD45R) surface receptor, which is expressed throughout the entire B lineage except plasma cells. Our protocol begins with a splenic perfusion technique,11 which yielded in six independent experiments an average of 1.58 (±0.236) x 108 spleen cells. It continues with MACS VS+ separation columns, which permitted us to recover from these single-cell suspensions 5.27 (±0.796) x 107 B220+ B cells (33.4% recovery) and 5.45 (±0.649) x 107 B220" non-B cells (34.5% recovery). One-third (32.1%) of the cells was typically lost. We refer the reader to the Web page of Miltenyi Biotec (www.miltenyibiotec.com) for detailed technical instructions on the fractionation of various cell types by MACS.

11 K. Felix, S. Lin, G. W. Bornkamm, and S. Janz, Eur. J. Immunol. 27, 2160 (1997).

pUR288 (placZ) Assay Shuttle Vector and Principle of Assay

In the plasmid-based shuttle vector pUR288, lacZ functions as both target and reporter gene of mutagenesis. The 5346-bp vector contains in addition to lacZ-coding sequences the 35-bp binding site for the LacI repressor, lacO, the binding site for CRP (c AMP receptor protein, which facilitates transcription of lacZ by stimulating the formation of an active promoter complex), an origin of replication (ori), and an ampicillin resistance gene (Fig. 4A). The principle of the pUR288

CRP binding site °IL/-lacO

CRP binding site °IL/-lacO

Amp 8 pUR288

lacZ

Hind III

Amp 8 pUR288

Hind III

lacZ

Amp tacZ H 1

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