50Jan Styczyński, Anna Jaworska(p
Quantitative analysis of changes in expression of leukemic markers during short-term prednisolone therapy in vitro 51Conditions of cell line culture <strong>and</strong> incubation ofleukemic cells. Cells were cultivated in culture mediumin incubator Forma Scientific Inc. model 3110(Marietta, Ohio, USA), in atmosphere of 5% CO 2 <strong>and</strong>temperature 37°C <strong>and</strong> humidity 90%. Cell culture wasperformed in culture flaks (Corning Incorporated,Corning, NY, USA, nr cat. 430639) with area 25 cm 2 .All experiments were prepared in laminar chamber(AURA 2000 M.A.C., Bioair Instruments, Opera, Italy).Quantitative analysis of CD10 antigen (Qifikit).Quantitative analysis of expression of CD10 antigenwas performed with the use of Qifikit (nr cat. K0078,DakoCytomation, Glostrup, Dania), based on nonstainedmouse monoclonal antibodies Anti-Human-CD10-CALLA (DakoCytomation, nr M0727), goatsecondary stained antibodies Goat-anti-Mouse Ig-FITC(DakoCytomation, nr F0479) <strong>and</strong> isotype controlMouse IgG1 (DakoCytomation, nr X0931) accordingto producer’s protocol (Figure 1).Flow cytometry. Analyses were done with flow cytometryEPICS XL (Coulter, Miami, FL, USA). Thisdevice is equipped with argon laser of power of 15mW, which causes fluorochrome excitation with wavelengthof 488 nm. These fluorochromes are detected ondetectors at following wavelengths: 515-535 nm (fluorescence1, FL1), 565-585 nm (FL2) <strong>and</strong> 610-630 nm(FL3). Cells were gated based on their FSC/SSC (forwardscatter, side scatter) signal. For each sample, allassays were performed twice: before <strong>and</strong> after 72 hoursof incubation with prednisolone.Immunophenotype analysis. Immunophenotypingwas performed according to guidelines of the NationalInstitute of Health, USA (Proteins Review On Web.Bethesda USA, http://www.ncbi.nlm.nih.gov/prow/guide/guide/45277084.htm), with the use of mousemonoclonal antibodies. Immunophenotyping was doneon isolated cell suspension. 25 µl of cell suspensionwas used, <strong>and</strong> 5 µl of monoclonal antibodies stainedwith fluorochrome was added. Cells were incubated inthe dark for 20 minutes, washed in PBS without calcium<strong>and</strong> magnesium, <strong>and</strong> then analyzed by flow cytometry.For each type of assay isotype control wasdone. The value of mean fluorescence intensity (MFI)was calculated as the difference between MFI of testedsample <strong>and</strong> MFI of isotype control. All patient sampleswere tested for the expression of following cell surfaceantigens: CD2, CD3, CD4, CD8, CD10, CD19 (TableI).Table I. Reagents, monoclonal antibodies <strong>and</strong> isotype controlsTabela I. Zastosowane odczynniki, przeciwciała i kontroleizotypoweReagentOdczynnikCD2-FITC/CD19-RPECD10-FITC/CD19-RPECD3-FITC/CD4-RPE/CD8-Cy5CD5-FITC/CD20-RPEProducerProducentCodeKodIsotype controlKontrola izotypowaAntibody cloneKlon przeciwciałDakoCytomation FR894 IgG1, κ MT910+HD37DakoCytomation FR883 IgG1, κ SS2/36+HD37DakoCytomation TC641 IgG1, κ DK25+MT310+UCHT1DakoCytomation FR729 IgG1, κ DK23+B-Ly1Fig. 1. Control histograms for quantitative analysis by Qifikit.(A) Negative <strong>and</strong> positive control. (B) Fluorescenceof st<strong>and</strong>ard beads enabling calibration lineformationRyc. 1. Histogramy kontrolne zestawu Qifikit. (A) Kontrolaujemna + kontrola dodatnia, (B) Fluorescencjawzorcowych kulek służących do określenia linii kalibracyjnejAB
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