We performed flow cytometry analysis on 100 μL of fresh blood from patients 2 and 4 and 10 healthy age-matched controls. We lysed the red blood cells in FACS Lysing Solution (BD Biosciences, Franklin Lakes, NJ) and stained all the samples with the following fluorescent antibodies: CD45 PerCP-Cy 5.5, CD55 PE, CD59 FITC, CD16 PE, CD24 PE (all from BD Biosciences), and with fluorescein-labeled proaerolysin (FLAER)-Alexa Fluor488 (Cedarlane, Burlington, ON, Canada), in accordance with manufacturers' instructions. We acquired the data by BD FACS Canto II Flow Cytometer and FACSDIVA software (BD Biosciences) and identified white blood cells by side scattered light (SSC) and forward scattered light. We selected granulocytes as granular (SSC-A high) and CD45-positive cells. For each marker, we measured the median fluorescence intensity (MFI) and calculated the ratio of patients' MFI vs the average MFI of controls. We used the Student t test to assess statistical significance of the MFI observations.
Cd59 fitc
Manufactured by BD
Sourced in Canada, Netherlands
CD59 FITC is a laboratory reagent used for the detection and identification of CD59 antigen, a glycosylphosphatidylinositol (GPI)-anchored cell surface protein, in various biological samples. It utilizes fluorescein isothiocyanate (FITC) as the fluorescent label to enable the visualization and quantification of CD59-expressing cells through flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cd55 pe, by BD (3 mentions)
Cd45 percp cy5, by BD (2 mentions)
Facsdiva, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Cd24 pe, by BD (1 mentions)
Cd16 pe, by BD (1 mentions)
Cd16 percp cy5, by BD (1 mentions)
Cd20 pe, by BD (1 mentions)
Cd19 alexa 700, by BD (1 mentions)
Cd56 pe, by BD (1 mentions)
Igd pe cy7, by BD (1 mentions)
Igm fitc, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
3 protocols using cd59 fitc
1
Flow Cytometry Analysis of Blood Cell Markers
2
Comprehensive B Cell Immunophenotyping
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LN tissue was put through a 70 μm (BD Falcon, San Jose, CA) cell strainer to obtain a single cell suspension. Cells were washed with PBS containing 0.01% NaN3 and 0.5% BSA and stained for 30 min at 4°C with directly labelled antibodies: CD45 PercP-Cy5.5, CD19 Alexa-700, CD27 APC-H7, IgD Pe-Cy7, CD20 PE, IgM FITC, CD69 PE, CD69 PerCP, CD21 APC, CD23 PE, CD25 APC, CD267 PE, BAFF-R FITC, CD16 Percp-Cy5.5, CD56 PE, CD55 PE, CD59 FITC (BD Biosciences, Breda, the Netherlands), HLA-DR Alexa-700 (eBioscience, Vienna, Austria), CD3 FITC (Sanquin, Amsterdam, the Netherlands). After incubation cells were washed and immediately analysed on a FACS CANTO II (BD Biosciences). To enable the measurement of different B cell subsets, a seven-colour FACS panel was set-up using antibodies against CD19, IgD, IgM, CD27, CD21, CD23 and CD45 (for normalization). Data were analysed using FlowJo software (Treestar, Ashland, OR, USA) and presented as frequencies, absolute numbers relative to 100 000 CD45+ lymphocytes or geometric mean fluorescence intensity (normalized on negative populations).
3
Flow Cytometric Analysis of GPI-AP Expression
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Fibroblasts derived from skin biopsies of patients, parents, and healthy control individuals were cultured in DMEM supplemented with 10% FCS, 1% ultraglutamine, 1% penicillin/streptomycin. For flow cytometry analysis confluently grown cells were washed twice with PBS (-Ca2+, -Mg2+); the cells were gently detached from the coulter dish with Trypsin-EDTA (0.01%). The single cell suspension was washed with FACS buffer, counted, diluted (100.000 cells/stain), and centrifuged, after which the supernatant was discarded and the cell pellet was resuspended in the following antibody mix.
Reduction of GPI-AP expression was calculated as a ratio between the median fluorescence intensity (MFI) of the patient against the mean of MFIs from healthy parents and a healthy unrelated control. It is noteworthy that heterozygous carriers of pathogenic mutations (parents) and unrelated healthy controls had only subtle differences in GPI-AP expression.
4 μl CD55-PE (BD #555694), 4 μl CD59-FITC (BD #555763), and 12 μl FACS buffer.
4 μl CD73-PE (BD#550257), 4 μl FLAER-AF488 (Cedarlane, FL2S-C), and 12 μl FACS buffer.
Reduction of GPI-AP expression was calculated as a ratio between the median fluorescence intensity (MFI) of the patient against the mean of MFIs from healthy parents and a healthy unrelated control. It is noteworthy that heterozygous carriers of pathogenic mutations (parents) and unrelated healthy controls had only subtle differences in GPI-AP expression.
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