Human Monocytes - CD14, CD16 - Ziegler-Heitbrock

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Handling of Mono Mac 6 Cell Line

Cell culture

Right after thawing a frozen sample, the 1 ml cell suspension is diluted to 10 ml with culture medium, cells are spun once, resuspended in culture medium and are seeded in 24 well plates at about 5 x10 5 /ml with 2ml per well and cultured over night.
Following this initial step, cells are seeded at a density of 2 x 10 5 /ml in 2 ml volumes in 24-well-plates and they are cultured at 37°C in a humidified 5% CO 2 atmosphere for 3-4 days before being used for experiments or before being pooled, counted and reseeded again (for washing spin at 400 g for 7 min). Doubling time is approx. 2 days. The culture medium is RPMI 1640 with 10-20 % fetal calf serum, fortified with several additives as detailed below.

The cultures have to be monitored on a regular basis for mycoplasma contamination and for the expression of the CD14 antigen (e.g. My4 antibody).

Upon receipt of the cell line, it is recommended to first establish a batch of frozen aliquots, that can be used to retrieve the original cell at regular time intervals or in case problems like contamination arise. For freezing of viable cells use 4-10 x 10 6 cells per ml in RPMI 20 % FCS and add dropwise an equal volume of RPMI 20 % FCS, 20 % DMSO (Merck, # 2931), while swirling the tube in ice water. For optimal freezing a controlled freezer device is recommended. Alternately, freezing may be tried by putting the tubes in a well insulated styrofoam box into a -80° C freezer overnight. Then transfer to liquid nitrogen (or storage at least at -135° C).

 

Composition of culture medium for Mono Mac 6

RPMI 1640 (Gibco or other)
L-Glutamine 2 mM (Gibco # 043-05030 H)

Penicillin 200 U/ml Streptomycin 200 ug/ml (Gibco#043-05140 H)

Non-essential amino acids (NEAA) 1-2 x (Gibco # 043-01140 H)

OPI-supplement Sigma 10 ml for 1 ltr ( Sigma # O - 5003)
contains oxalacetic acid, sodium pyruvate and insulin

filtration to remove LPS (see below), then add fetal calf serum 10-20%, low LPS

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THE COMMERCIAL SOURCES GIVEN ARE THOSE USED IN OUR LABORATORY, BUT TISSUE CULTURE GRADE REAGENTS FROM OTHER SUPPLIERS CAN BE EXPECTED TO WORK AS WELL
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Removal of LPS

In case of gross contamination of culture medium with lipopolysaccharide, the Mono Mac 6 cells will grow slower and will clump. A typical pattern when inspecting the 24 well plate under the inverted microscope looks as follows:


    no LPS           LPS (e.g. 10 nanog/ml)

In order to avoid introduction of LPS, use quality reagents only. If required, LPS may be removed by ultrafiltration using a 20 kD cut off column (Ultra Steri Set, Cat. No.: 8394189, Gambro Medizintechnik GmbH or USA: COBE Laboratories Inc.).

Gambro Medizintechnik GmbH
Lochhamer Str. 15
D-82152 Planegg-Martinsried
Germany

Tel: +49 89 899 330


BCT Laboratories Inc.
Lakewood, Colorado
80215-4498
USA

Tel: +1 303 232 6800 or +1 877 339 4BCT


For this procedure, fetal calf serum (> 20 kD) has to be added after filtration.
In case you are not interested in LPS induced gene expression you may generate a LPS resistent line by feeding the cells with gradually increasing doses of LPS (Salmonella minnesota, L-6261, Sigma) from 0.5 ng/ml to 1 ug /ml which can be done within 3 weeks (see below). You then always supplement your culture medium with 1 ug LPS / ml.

Absence of LPS in a batch of culture medium may be verified by the Limulus assay or by the cell clumping in 24 well plates (see Fig.).

Mycoplasma testing and removal

As with any other cell culture a contamination with mycoplasma can cause major problems in phenotypic and functional properties of the Mono Mac 6 cell line. Regular screening for absence of mycoplasma is recommended. Mycoplasma can be removed by 2 cycles of 3h incubation with 10% non -heat-inactivated human serum followed by cloning of the cells. Also contamination may be avoided by including 5% non -heat-inactivated human serum in the regular culture medium. Complement in human serum will detach mycoplasma from the cell surface and will then kill the microorganisms.

Establishment of a LPS resistant subline

In case you want to in obtain a LPS resistant Mono Mac 6 cell line you may generate it by adding gradually increasing doses of LPS to a culture. A possible scheme that takes less than 3 weeks of time is the following.
4 wells of Mono Mac6 cells are started at 4 x 10 5 /2 ml per well with LPS at 0.5 nanog/ml. Cells are harvested after 2-3 days adjusted to 4 x 10 5 /2 ml and reseeded with LPS at 2 nanog/ml. This is repeated every 2-3 days with the following increasing doses of LPS: 5, 20, 50, 100, 200, 500 and 1000 nanog/ml.
Cells can then be kept continously in culture medium supplemented with the 1000 nanog/ml dose of LPS. The advantage of having an LPS resistent variant are:
a.) in case you work on bacteria-monocyte-interaction you may dissect the effects of LPS and those exerted by other bacterial components.
b.) in case you want to entirely disregard LPS, you may, by continously feeding the cells with LPS, avoid the production of LPS free (or low) culture medium as is required for the standard Mono Mac 6 cell line.

Please note that the properties of LPS resistant Mono Mac 6 cells have not been studied in detail. A side-by-side comparison with the maternal line may therefore be informative.

References

  1. Ziegler-Heitbrock HWL, Thiel E, Fuetterer A, Herzog V, Wirtz A, Riethmueller G: Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int. J. Cancer 41: 456, 1988.
  2. Ziegler-Heitbrock HWL, Burger R: Rapid removal of mycoplasma from cell lines mediated by a direct effect of complement. Exp. Cell Res. 173: 388, 1987.