Human Monocytes - CD14, CD16 - Ziegler-Heitbrock

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Mycoplasma-Screening using the Indicator Culture

Indicator cells

are mycoplasma-negative Vero B4 cells that are grown as adherent cells in tissue culture flasks in RPMI 1640 medium with 10% fetal calf serum, Penicillin, Streptomycin and l-Glutamine. Cultures are harvested once a week using EDTA at 1mM and are reseeded at 1 : 30 dilution.

Set up in chamber slides

is done routinely on a Thursday with Vero B4 cells adjusted to 5 x 10 3 cells / ml and 400 µl volumes each are given into the 8 chambers of Lab-Tek chamber slides (#177402, Nunc, Naperville, Il, USA).

Addition of test supernatant

is routinely done on a Friday in that 100 µl of cell culture supernatant to be tested is added. The test culture supernatant should be taken from cultures grown to high cell density. It can be collected during the week and is stored at 4°C for up to 5 days before addition to the chamber slides. As a positive control we use aliquots of -80°C stored positive culture supernatant, as a negative control we use the complete culture medium.

Testing for mycoplasma

is then done on a Monday. Chambers are rinsed 3 times with Hanks' buffer, then 100% Methanol is added to each chamber and the cells are fixed for 5 min at RT. Then chambers are rinsed 3 times with Hanks´s and 100 µl of Hoechst 33258 dye at 1 nanog/ml in H 2 O is added for 15 min at 37°C.

(A stock of Hoechst 33258 is stored at 1 microg/ml in H 2 O in a light protected tube at 4°C). Chambers are then again rinsed with Hanks 3 times. The chamber frame and the gasket is removed and and the slide is covered with glycerol-buffer (2.33g Citric acid, #244, Merck, 4.12 g Na 2 HPO 4 , 250 ml of Glycerol, 250 ml of H 2 O). The slide is covered with a large cover slip and is inspected under a UV-microscope. Take care to keep the cells wet at all times.

Positive cultures show a speckled staining on the surface of the Vero B4 cells (left) while negative samples only show staining of the Vero B4 nuclei and sometimes some nuclear fragments in the cytoplasm (right).

Reference: Ziegler-Heitbrock H.W.L. & Burger R.: Rapid removal of mycoplasma from cell lines mediated by a direct effect of complement. Experimental Cell Research, 173: 388-394, 1987.