TABLE 1.

Modified caspase-3 assay for serum neutralization of macrophage cytotoxicity

Assay stage	Step	Descriptiona	Comment
Cell and bacterial culture	1	Seed J774.A1 MΦs into 96-well trays (200 μl/well) of a 5 × 105 cell/ml suspension.	Use freshly prepared EMEM with Earle's salts (Invitrogen) supplemented with 2 mM nonessential amino acids (Sigma), 2 mM l-glutamine (HyClone), and 10% FBS (Gibco).b
			The J774.A1 MΦs performed optimally in the assay when used at a passage less than 25; cells passaged at <22 were used.
	2	Inoculate an overnight culture of Yersinia pseudotuberculosis strain Yptb pTrcV (1) in BHI broth supplemented with ampicillin, kanamycin, EDTA, and MgCl2, as described previously (1).	Use fresh growth from a BHI agar slant consisting of BHI agar with ampicillin and kanamycin.
	3	Dilute the overnight culture 1/20 into 25 ml of BHI broth and incubate the flask at 37°C for 2 h with shaking at 225 to 250 rpm.
Preparation of bacterial inoculum	4a	After the culture has incubated for 2 h, transfer the culture to a 50-ml centrifuge tube and centrifuge the culture for 10 min at 2,600 rpm to sediment the bacteria.	Vigorous growth of the bacteria during the 2-h preculture step is important; cultures that do not increase at an OD620 of at least six- to eightfold
	4b	After centrifugation is complete, resuspend the pellet in 3 ml of EMEM-FBS and determine the bacterial concn (with, e.g., an Ultrospec UV spectrophotometer; Amersham Pharmacia) at OD620. Dilute and adjust the bacterial inoculum in warm EMEM-FBS to an OD620 of 0.25 in the required vol (see step 6).	(3-4 doublings) are often not sufficiently cytotoxic.
Pretreatment	5	Prepare the J774 tray. Just before treating the bacteria with anti-V Ab/serum, remove the medium and immediately add prewarmed EMEM-FBS to the 96-well trays (200 μl of EMEM-FBS in uninfected control wells and 100 μl in the well to be infected).
	6a	Pretreat the bacteria and add the sample to the tray. For each sample, combine 0.5 ml of adjusted bacteria in a microtube with Ab/serum (in amts of 10-40 μl) diluted to twice that of the final desired dilution in the tray. Use medium instead of Ab for the negative control sample. For titration experiments, the Ab/serum is prediluted in EMEM-FBS in a microtiter tray, and 20 μl of each is added to a tube of bacteria.
	6b	Incubate the microtubes at 38°C for 5 min with shaking (1, 9) and add 100 μl of the pretreated bacteria to the wells that are to be infected. Test all samples in quadruplicate.
Incubation and development	7	Centrifuge the tray for 5 min at 800 rpm in a centrifuge that accommodates 96-well-plate carriers.
	8	Incubate the tray for 2 h at 37°C in 5% CO2. Dilute gentamicin (Invitrogen) into EMEM-FBS and add 20 μl of the dilution to each well, for a final concn of 50 μg/ml gentamicin.
	9	Incubate the tray once more for 1.5 h, remove the supernatants from the wells, and wash the samples once, gently, in 10-20 mM PBS without magnesium or calcium.
	10	Remove the medium and measure caspase-3 levels as described in the EnzChek caspase-3 II kit instructions (Molecular Probes).
		After adding the lysis buffer, place the tray in a −70°C freezer until the medium is frozen, and then thaw the medium completely by placing the tray in a 37°C incubator.
		Centrifuge the tray and transfer the supernatants to a black, clear bottom tray (Costar). Add an equal volume of reaction solution and, after incubating the sample for 30 min, read the fluorescence with, e.g., Softmax Pro software on a Gemini or M2e spectrometer (Molecular Devices) with settings of 485 nm em and 530 nm ex. Export the data to an Excel spreadsheet for regression analyses (see text).

^aAbbreviations: EMEM, Eagle's minimal essential medium; FBS, fetal bovine serum; BHI, brain heart infusion; OD₆₂₀, optical density measured at 620 nm; Ab, antibody; Ab/serum, antibody or serum; em, emission; ex, excitation.

^bThe complete medium, containing the components listed here, is referred to herein as EMEM-FBS.