Danish Society for Flow Cytometry

25th Meeting - Wednesday, April 25, 2001


Functional Assays in Cytometry

Auditorium 2, Rigshospitalet, Blegdamsvej 9, Copenhagen

Organizers: Carl-Henrik Brogren, Lars Ryder and Jørgen K. Larsen

Sponsors: The Danish Medical Society and RAMCON A/S (Beckman Coulter)

All are welcome!


Session I (Chair: Carl-Henrik Brogren, DSFCM)

13:00-13:50      Functional characterization of phagocytic cells in acute and chronic inflammation

                        Stefan Barlage, Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany

13:45-14:05      Phagocytic activity of pulmonary macrophages assessed by flow cytometry

                        Lars Peter Nielsen, Institute of Pharmacology, Center of Clinical Pharmacology, University of Aarhus

14:05-14:15      Phagocytosis and oxydative burst studied in multicolor cytometry

                        Ana Aiastui Pujana, Susana Alvarez Herrero and Carl-Henrik Brogren, Division of Microbiology, Institute of Food Safety and Toxicology, Ministry of Food, Agriculture and Fisheries, Søborg

14:15-14:35      Pause - Refreshments

                        Session II (Chair: Lars Ryder, Dept. of Clinical Immunology, Rigshospitalet, Copenhagen)

14:35-15:15      Functional studies in human whole blood monocytes

                        Marta Borg, AstraZeneca R&D, Lund, Sweden

15:15-15:35      Respiratory chain activity of VBNC-Campylobacter studied by flow cytometry

                        Birthe Hald, Dept. of Poultry, Fish and Fur Animals, Danish Veterinary Laboratory, Aarhus

15:35-16:00      Pause

16:00 -             General assembly of the Danish Society for Flow cytometry


President          Jørgen K. Larsen, Finsen Laboratory, Finsen Center, Rigshospitalet, Dept. 8621, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark. Tel +45 3545 5751. Fax +45 3538 5450. E-mail j.k.larsen@finsenlab.dk.

Vice-president   Jens Peter Stenvang, DAKO A/S, Produktionsvej 42, DK-2600 Glostrup, Denmark.. Tel +45 4485 9500. Fax +45 4485 8435. E-mail jenspeter.stenvang@dako.dk.

Meetings coordinator  Carl-Henrik Brogren, Division of Microbiology, Institute of Food Safety and Toxicology, Ministry of Food, Agriculture and Fisheries, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark. Tel +45 3395 6184.  Fax +45 3395 6001. E-mail chb@fdir.dk.

Secretary          Hans Jürgen Hoffmann, Department of Respiratory Diseases, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. Tel +45 8949 2107. Fax +45 8949 2110. E-mail dsfcm@dadlnet.dk or hansjuergen.hoffmann@get2net.dk.

Treasurer          Bjarne Møller, Department of Clinical Immunology, Odense University Hospital, DK-5000 Odense C, Denmark. Tel +45 6541 3576. Fax +45 6612 7975. E-mail bjarne.moeller@ouh.dk.




Functional studies in human whole blood monocytes

Stefan Barlage, Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany

Chemokines are a family of pro-inflammatory activation-inducible cytokines that are implicated in inflammation and in the recruitment of various celltypes. They are divided into four sub-families (CXC, CC, XC, CX3C) and today it has been identified fifty chemokines and twenty chemokine receptors. These receptors form a structrually related group within the superfamily of G-protein-coupled receptors which mediate signalling via heterodimeric G-proteins.

Chemokines and their receptors are not only essential mediators of normal leukocyte trafficking but they are also multipotent cytokines that localize and enhance inflammation by inducing chemotaxis and cell activation of different types of inflammatory cells present at sites of inflammation. Chemokines have been shown to exert their effect on distinct subsets of cells. CXC-Chemokines, for example appear to attract neutrophils but not macrophages, while CC-Chemokines preferentially induce migration of macrophgages and T cells.

Chemokines binding to chemokine receptor induces second messenger realise (Ca2+), gene transcription and cytoskeletal rearrangement.

We have investigated the functional activity of CC chemokine receptors in human whole blood monocytes using FCM based ratiometric Ca2+ flux, measurement of CD11b upregulation and measurement of the rearrangement in the actin cytoskeleton by Phalloidin-A.


Phagocytic activity of pulmonary macrophages assessed by flow cytometry.

Lars Peter Nielsen, MD. Center of Clinical Pharmacology, University of Aarhus.

Bronchoalveolar lavage (BAL) is a well-recognized method to provide cells from the lower airways for analysis. In general, the cellular constituents of BAL fluid are, almost exclusively, made up of alveolar macrophages (AM) and lymphocytes. The two cell types are easily distinguished on the basis of size and granularity by flowcytometry, ie. forward and side scatters. Antigen presenting cells, eg. AM, hold a key position in activating the immune system. Examining the phagocytic proces in AM could thus be important in various immunologically related lung diseases, eg. asthma, sarcoidosis and idiopathic pulmonary fibrosis.

We studied the phagocytic proces in AM from patients undergoing BAL for diagnostic purposes. After adjustment for viability, 2.5 x105 cells were incubated with FITC-labeled yeast particles, at body temperature, for time periods ranging from 0 to 120 minutes. The increase in fluorescense intensity, when gating for AMs, were then used as an expression of their phagocytic activity.

Fifty-seven consecutive patients suffering various lung diseases, the most frequent being sarcoidosis (N=10), lung fibrosis (N=9) and chronic obstructive lung disease (N=9), were included. No significant correlations between age, sex or disease and AM phagocytic activity were seen. AM from non-smokers demonstrated significantly higher phagocytic activity compared to AM from smokers (p<0.05). Moreover, a negative correlation was observed between tobacco consumption and AM phagocytic activity (r=-0.51, p<0.05). A significant difference in phagocytic activity was also found between incubation buffers, ie. HBSS and RPMI, relating to their buffer capacity, and favouring the latter (p<0.01).

In conclusion, the phagocytic activity of AM is highly dependent upon pH of the environment. The phagocytic activity of AM is reduced by smoking and the degree of reduction is correlated to tobacco consumption.


Respiratory chain activity of "VBNC"-Campylobacter studied by flow cytometry

Birthe Hald & Mogens Madsen, Danish Veterinary Laboratory, Dep. of Poultry, Fish, and Fur animals, Århus.

The tetrazolium salt CTC (5-Cyano-2,3-ditolyl tetrazolium chloride) has previously been used as a viability detector in microbiology due to its ability to form intracellular granula of CTF (red flourescent formazan) when it is reduced by an active respiratory chain. With the aim of predicting the chance of resuscitation in 32 campylobacter microcosms (~108/ml Campylobacter jejuni), we have used CTC and flow cytometry to quantify the viable potential of the populations.

The following protocol was found adequate to form intracellular CTF granules of approx. 0.2-1.5m . One ml of microcosm was centrifuged (8000 g, 5 min), the pellet resuspended in 1 ml sterile water at 2° C, incubated at 37° C with 5.5 mM Formate and 0.5 mM CTC for 1 h, and formaldehyd added at 2%. CTF precipitations were recorded on an Epics XL flow cytometer. The flow protocol was set up to record the precipitations by log FS, log SS, and log Fl 3, with discriminator setting on FL 3 to exclude the non-flourescent CTF negative bacteria. WinMDI 2.5 was used for off line data analysis. Gating was performed on logFS/logSS scattergram with usually 95-99% of the events lying within the gate, and counts obtained as FL3 positive events.

In newly launched microcosms, the number of respiratory active campylobacters were exceeding the colony forming units (CFU) with ~1 log(10)unit pr. ml. At the time of non-culturability of the populations, 104 - 106 campylobacter/ml (0,01 - 1%) were still able to reduce CTC. Despite the high rate of respiratory and enzymatic activity that is reflected by the number of CTC positive C. jejuni in the microcosms, no resuscitation experiment at all succeeded, neither by in vitro experiments nor by infection experiments with day-old chicks.

We found the recording of the CTF-precipitations by flow cytometry useful and reliable; however concluded, that the recorded respiratory activity was not synonymous with viability of C. jejuni.