Pulsing macrophages with suspension Equipment and reagents

• 4 ml polypropylene culture tubes (Falcon 2063)

• Centrifuge equipped with SorvalHlOOOB rotor

• BMM0 macrophages obtained as described in Protocols 1 and 2, or other macrophages as described in Chapter 1

Method

1 Resuspend macrophages at 106/ml.

2 Aliquot 3 ml into replicate polypropylene culture tubes and keep on ice until use.

3 Add bacteria/protozoa at varying multiplicities of infection (MOI) in 500 n-1 complete tissue culture medium.

4 Pellet microbes and macrophages together by centrifugation at 2000-3500 r.p.m. at 4°C.i

5 Warm tubes to 37°C by placing in a water-bath.

particulate antigens In

• Complete tissue culture medium (see Protocol 3)

• 1% (w/v) paraformaldehyde in PBS {see Protocol 3)

Protocol 7 continued

6 Incubate for varying periods of time (typically from 15 min to up to 24 h).

7 Remove a tube at each time point and chill on ice,

8 Remove the majority of non-ingested micro-organisms by resuspending pellet and differential centrifugation at 1000 r.p.m. for 10 min at 4°C.b Discard supernatants, to appropriate disinfectant if pathogens are involved,

9 Fix macrophages with 1% paraformaldehyde as described in Protocol 3, using 500 of fixative,

10 Use fixed macrophages as APC source in desired functional assay.

•*The required centrifugation will be dependent upon the micro-organism in question rather than the macrophage population. This centrifugation step allows rapid intimate contact between macrophage and microbe, and helps to ensure a uniform rate of uptake. " If organisms cannot be removed efficiently by two to three rounds of centrifugation then proceed directly to step 9. However, excess free organisms may influence later T cell function or be subsequently processed by contaminant APCs in the T cell preparation.

3.2.2 Assessment of infection ievel/antigen dose

Given the above discussion, it is particularly important to ascertain the actual presentable antigen dose within macrophages exposed to infectious agents. The methodology for accomplishing this will vary depending on the organism, and may involve:

(a) Direct staining of macrophage monolayers or cylospins.

(b) Lysis of macrophages, followed by re-culture and counting of released organisms (by limiting dilution, [:,H]TdR uptake, or other biochemical means).

(c) The use of flow cytometry.

In the latter instance, we have found that use of the CFSE and CMFDA Cell Tracker dyes (Molecular Probes Inc.) provide a convenient means of pre-labelling protozoan and bacterial pathogens. However, it should be noted that as these are vital stains and require metabolic activity to yield a fluorescent product, they do not indicate the degree of contamination of the microbe population with dead organisms (see Scction 3.2,3). We have therefore found that flow cytometric methods are limited to assessing the extent that infection alters phenotype, rather than as a definitive means of evaluating antigen uptake. Protocol 8 outlines a rapid procedure for histochemical assessment of microbe uptake by macrophages.

When adherent monolayers are used, the importance of substrate in determining adhesive properties can not be underestimated (see also Chapter 2). Although it may be tempting to adhere macrophages to glass covers lips to enumerate pathogen uptake, this may not reflect directly the status of a macrophage monolayer which has adhered to tissue culture plastic and then been washed several times before addition of T cells.

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