Microglia by Unknown

Author:Unknown
Language: eng
Format: epub
ISBN: 9781493996582
Publisher: Springer New York

2.Trypanosome culture medium: HMI-9 medium (see Subheading 2.1).

3.Antibodies for microglia labeling (see Subheading 2.4).

4.Labeling buffer-HEPES plus (see Subheading 2.4).

5.Low-binding Eppendorf tubes.

6.24-well plates.

7.CO2 incubator, refrigerated centrifuge.

3 Methods

3.1 Cultivation of T. brucei Bloodstream Form

In accordance with the nature of the parasite to be analyzed, it is important to make sure that cells are completely healthy before starting the experiment. In our assessment, we analyzed interactions between microglia and bloodstream forms of T. brucei; however, the principle of this technique can be applied for any extracellular protozoan parasite. Consequently, we describe here the cultivation of bloodstream forms of T. brucei and those genetic manipulations of the parasite that are necessary to perform the assay.

Trypanosomes can be maintained in culture for long periods of time by dilution with fresh medium at defined intervals. Parasites are cultivated in HMI-9 medium containing 10% inactivated fetal bovine serum (FBS) and penicillin–streptomycin under a 5% CO2 atmosphere at 37 °C. In these experiments, the parasite strain SMB was used. It requires Geneticin as selective marker; therefore, this antibiotic is a regular constituent of the culture medium (see Subheading 3.2).1.Depending on the availability, trypanosomes culture can be prepared either from a previous liquid culture during the exponential growth phase or from a stabilate stored in liquid nitrogen (see Note 1 ). In the former case, determine cell density of trypanosome culture by direct counting in a hemocytometer. Make sure the suspension is homogeneous before loading the chamber. Only motile trypanosomes are considered viable cells. Proceed with step 2. In the latter case, before counting, frozen parasites have to be handled as follows: take an aliquot of the parasite suspension out from the liquid nitrogen tank. Defrost cells quickly in a 37 °C water bath. Resuspend parasites in 9.5 mL trypanosome culture medium and centrifuge at 800 × g for 10 min to wash out the DMSO contained in the freezing medium. Discard the supernatant, resuspend parasites in 0.5 mL of fresh medium, and count the cells in a hemocytometer (see Note 2 ).

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