A comprehensive immunophenotyping panel was performed blinded to treatment and outcome at a median of 28 days pre-AHCT (N=104) and at Day +100 post-AHCT (N=83), via methods previously described [14 (link)]. Blood for flow cytometric studies was drawn into heparinized tubes and processed by the laboratory within 24 hours of collection. Briefly, blood was washed 3 times with flow cytometry buffer (0.5% BSA, 0.04 g/L Na2EDTA and 0.1% sodium azide in PBS pH 7.2) and immunophenotyped using a stain-and-then-lyse technique. Data acquisition was performed on a FACSCanto II flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA) controlled daily using cytometer, setup and tracking beads. For the peripheral blood phenotyping, 50 000 events were acquired for each of the determinations. Data were analyzed using WinList version 6 (Verity Software House, Topsham, ME). The comprehensive flow cytometry strategy to identify the different lymphoid subsets is shown in Table 1 .
Winlist version 6
Manufactured by Verity Software House
WinList version 6 is a software product developed by Verity Software House for the analysis and visualization of flow cytometry data. It provides a comprehensive suite of tools for the processing, gating, and presentation of flow cytometry results.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using winlist version 6
1
Comprehensive Immunophenotyping for Autologous HSCT
2
Comprehensive Immunophenotyping for Autologous HSCT
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A comprehensive immunophenotyping panel was performed blinded to treatment and outcome at a median of 28 days pre-AHCT (N=104) and at Day +100 post-AHCT (N=83), via methods previously described [14 (link)]. Blood for flow cytometric studies was drawn into heparinized tubes and processed by the laboratory within 24 hours of collection. Briefly, blood was washed 3 times with flow cytometry buffer (0.5% BSA, 0.04 g/L Na2EDTA and 0.1% sodium azide in PBS pH 7.2) and immunophenotyped using a stain-and-then-lyse technique. Data acquisition was performed on a FACSCanto II flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA) controlled daily using cytometer, setup and tracking beads. For the peripheral blood phenotyping, 50 000 events were acquired for each of the determinations. Data were analyzed using WinList version 6 (Verity Software House, Topsham, ME). The comprehensive flow cytometry strategy to identify the different lymphoid subsets is shown in Table 1 .
3
Immunolabeling and Flow Cytometry of Plasmodium falciparum
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The 3D7 P. falciparum clone and one Ghanaian patient isolate (BM057) were cultured in vitro (26 (link)) and were selected for DC4 PfEMP1 IE surface expression by repeated Ab selection as described previously (12 (link)). The identity of the isolates was routinely verified by genotyping as described previously (27 (link)), and Mycoplasma infection was regularly excluded using the MycoAlert Mycoplasma Detection Kit (Lonza) according to the manufacturer’s instructions.
P. falciparum IE were DNA-labeled with ethidium bromide and surface-labeled with mouse antisera obtained from the immunized mouse used for hybridoma production (15 μl serum/well), 24E9 mAb (100 μg/ml), or 24E9 Fab fragments (100 μg/ml). Whole Abs were labeled using an FITC-conjugated secondary anti-mouse IgG (1:100; Vector Labs), and an anti-mouse F(ab′)2 IgG (1:100; Jackson Immunoresearch) was used to detect Fab fragments. FITC fluorescence data from ethidium bromide+ cells were collected on a Cytomics FC 500 MPL flow cytometer (Beckman Coulter) and analyzed in WinList version 6.0 (Verity Software House).
P. falciparum IE were DNA-labeled with ethidium bromide and surface-labeled with mouse antisera obtained from the immunized mouse used for hybridoma production (15 μl serum/well), 24E9 mAb (100 μg/ml), or 24E9 Fab fragments (100 μg/ml). Whole Abs were labeled using an FITC-conjugated secondary anti-mouse IgG (1:100; Vector Labs), and an anti-mouse F(ab′)2 IgG (1:100; Jackson Immunoresearch) was used to detect Fab fragments. FITC fluorescence data from ethidium bromide+ cells were collected on a Cytomics FC 500 MPL flow cytometer (Beckman Coulter) and analyzed in WinList version 6.0 (Verity Software House).
4
Characterizing PfEMP1 Surface Expression
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