**2. Aim**

**2.1** Comparison of the effects of Viscum album QuFrF extract with those of Vincristine.

**2.2** Mode of action of Viscum album QuFrF extract and Vincristine.

**2.3** To assess the effective doses of Viscum album QuFrF extract and to transfer these doses to the in vivo situation.

The Effects of *Viscum album* (Mistletoe) QuFrF Extract and Vincristine in Human Multiple

**3.7 Measurement of membrane expression of IL-6 receptor** 

**3.6 Measurement of cytokine production** 

**3.8 Measurement of the cell cycle phases** 

**3.9 Measurement of apoptosis and necrosis** 

percent of total cell number.

**3.10 Measurement of the proliferation** 

the untreated cells was <10% (3–8%).

**3.11 Statistical analysis** 

P<0.05.

pg/ml or 5 pg/ml.

were taken as 100%.

(100%).

Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters 591

The IL-6 production or IL-10 production in the supernatant of the cultured cells was determined by chemiluminescent immunometric assay. The lowest detectable level was 2

For immunofluorescence staining 3x105 cells/100 l were incubated with 20 l phycoerithrin (PE) conjugated monoclonal antibody (CD 126, Immunotech, France) for 30 min at 4 ◦C. Then the cells were washed, sedimented and analysed in the FACSCalibur flow cytometer. For the expression of the membrane IL-6R (CD 126) the signal intensity (geometric mean of the fluorescence intensity x counts) was used as parameter. The signal intensity of the treated samples was compared with that of untreated samples, which

The cell cycle phases GO/GI, S, G2/M were assessed using the cycle test plus DNA reagent kit on a flow cytometer (BD, BioSciences, San Jose, USA No 340242). Briefly: 5x10**5** cells were incubated at room temperature with trypsin buffer and additionally with trypsin inhibitor+RNAse buffer. The values are expressed in percentage of total viable cell number

Apoptosis was measured using Annexin V-FITC (BD Biosciences Pharmingen, San Diego, USA No 556 570). Necrosis was measured using propidium iodide (PI). Briefly: 1x105 cells were incubated with Annexin V-FITC or PI at room temperature in the dark. Thereafter the samples were analysed in a flow cytometer. Apoptotic cells: Annexin V-FITC positive and PI negative. Necrotic cells: Annexin V-FITC positive and PI positive. The values are given in

The proliferation was assessed using cell proliferation reagent WST-1 (Roche, Mannheim, Germany, No 1644 807). The colorimetric assay is based on the reduction of the tetrazolium salt WST-1 by viable cells. The reaction produces the soluble formazan salt. The quantity of the formazan dye is directly correlated to the number of the metabolically active cells. The proliferation rate was measured 1, 2 and 4 h after incubation with the reagents at time points 24, 48 and 72 h. The upper limit of the absorbance was 2.0–2.1. The intra-sample variance of

Three to four independent measurements were carried out. For the evaluation of the parameters the Mann-Whitney U-test was used. The limit of significance was taken as
