THREE TIPS IN METHODOLOGY
 
GIUSEPPE BASSO
UNIVERSITY OF TORINO
DEPARTMENT OF PEDIATRICS
Piazza Polonia, 94
Torino
tel. +39 11 3531204
fax +39 49 821.3510
E-mail: gbasso@child.pedi.unipd.it
 
MULTIDRUG RESISTANCE ASSAY

Several proteins are involved in multi drug resistance which act as an efflux pump; MDR-1,MDR-3,MRP,LRP.....Several distinct classes of chemiotherapeutic agents are substrates for these proteins and can be actively transported out of cells or compartmentalisated into the cells.These proteins can be studied or by their expression using specific monoclonal antibodies or by functional assay.

The functional assay is completely different from antigens expression .
 

GENERAL PROCEDURE

Resistant Cell lines .

Colon adenocarcinoma cell line LOVO109 and its MDR doxorubicin (Dx) selected subline LOVO Dx and T-cell acute lymphoid leukemia cell line CEM and its MDR vinblastina (VBL) selected subline CEM-VBL or equivalent cell lines are used as control. The cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum 2mM glutamine, 50ugr/ml penicillin and 50ugr/ml streptomycin in 5% CO2. LOVO Dx cells were grown in presence of 100ngr/ml doxorubicine, CEM-VBL in the presence of 10ugr/ml vinblastine.

Clinical samples.

1x106 cells in 1ml medium obtained from surgical biopsies, mechanical (not chemical) desegregated, or peripheral blood or bone marrow were used.

Viability > 90% , if not the blasts viable cells were isolated by density gradient centrifugation . Tumor cells or mononuclear cells, removed from the plasma-ficoll interface were washed three times in RPMI.

Rhodamine-123 efflux (RHD-E).

Efflux of Rhd-123 (Sigma) was studied in 1x106 cells/ml.
Stain 2 aliquots of cells in medium ( RPMI-1640 plus 10% fetal calf serum) with 200ngr/ml Rhd-123 for 15 minutes at 37°C , 1 tube without and 1 tube with inhibitors ( Cyclosporin A Sandoz at 0,4 uM, or Verapamil (10uM) Knoll, or PSC 833 Sandoz at 1,6uM ).
Wash in ice cold staining medium and resuspending in dye free medium in 4 different tubes.
One tube in ice is the control at time 0
The cells efflux dye were allowed for 1 hour in dye-free medium at 37°C (at time 15, 30, 60 min.) in the presence or absence of MDR inhibitors.
Time dependent accumulation/efflux, expressed as maximum mean Rhd-123 fluorescence channel has been determinate.

Daunorubicin (DNR)efflux

The experiments were performed also with staining with DNR (4ugr/ml) for the comparison of Rhd-123. The cells were stained for 15 minutes at 37°C with or without MDR inhibitors, washed , and analyzed at the same time intervals (time 0, 15, 30 and 60 minutes).

Flow-cytometric analysis.

Cells were analyzed on EPICS XL (Coulter, Hialeah, FL) operated at 488nm which detect green (Rhd-123) and red (PE/DNR) fluorescence. Rhd-123 fluorescence was collected through a 530/30 nm and PE/DNR fluorescence through 585/40nm band pass filter.The overlapping emission spectra were electronically compensated. Data acquisition and analysis were performed with the specific software. Forward and side scatter signals were collected using linear scales; leukemic cells were gated according to these parameters, further confirmed by an extensive diagnostic antibody panel used for leukemia immunophenoptype. Fluorescence signals were collected on a logharitmic scale. In not homogenous samples, the double staining would be performed by PE direct conjugate primary antibody (immunological gate) and by Rhd-123 (the compensation is 30-35%).

Dynamic evaluation of RhD-123 assay

A continuos evaluation of accumulation/efflux of Rhd-123 in function of time are determinate.
1x106 cells were run in flow cytometer in basal condition to control the fluorescence.
After 2-3 min. 200ngr Rhd-123 are included in the tube without break off the acquisition .
This test evidenced a stable pattern of fluorescence for the non MDR cells during 30 minutes of observation .The evaluation MDR+ cell lines showed, on the other hand, an oscillating fluorescence pattern indicating a high transport-pump activity, with accumulation and efflux in medium with excess Rhd-123 .
 


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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone:(765) 494-0757; FAX (765) 494-0517; Web http://www.cyto.purdue.edu, EMAIL cdrom3@flowcyt.cyto.purdue.edu