Harvested cells (about 50000 cells) were incubated with cold PBS before staining with Abs, and then cells incubated with the cell-surface Fc blocker receptors antibody (PharMingen, San Diego, CA, USA) for 15 min. Then after centrifugation the cells were incubated with PE-conjugated anti-rat c-kit, (c-Kit (2B8): sc-19619, USA), and FITC-conjugated anti-rat FcɛRI antibody (BD Pharmingen: No.551469, USA) for 30 minutes at 4°C. In parallel isotype-matched non-specific Abs were used as the background staining control. Live events were acquired on a FACSCalibour flow cytometer (BD Biosciences), and data were analyzed with FACSDiva software (v6.1.2) (19 (link)).
Facscalibour flow cytometer
Manufactured by BD
Sourced in United States
The FACSCalibour is a flow cytometer designed for multi-parameter analysis of cells and particles. It utilizes laser-based detection and fluidics technology to measure and analyze the physical and fluorescent characteristics of individual cells or particles suspended in a fluid stream.
Automatically generated - may contain errors
6 protocols using facscalibour flow cytometer
1
Flow Cytometry Analysis of Mast Cell Markers
2
Flow Cytometric Analysis of HLA-G Expression
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After this period, the cells were swept from the bottom of the wells by trypsin and washed in PBS. Treated cells and control cells were suspended in 1 ml PBS and then, 1 µL of monoclonal anti HLA-G-FITC/MEMG-09 (Exbio) were added into tubes. These tubes were incubated for 30 min in the dark and at 4℃. Also, isotypic control cell was prepared by adding 1 µL mouse IgG1 isotype control-FITC (Exbio) to untreated cell suspension in 1 ml PBS to confirm the specificity of primary antibody binding. After twice washing by wash solution, ADSCs were ready for analyzing at FACS Calibour flow cytometer (BD) and 10000 events were counted and then the gating of wanted cell population eliminated dead cells and debris. In the next step, the fluorescence intensity of gated cells was analyzed by Cell Quest Software program.
3
Apoptosis Induction in Bel-7402 Cells
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For apoptosis identification, 2 × 105 log-phase Bel-7402 cells were seeded in poly-HEMA coated 6-well plate. On the second day, cells were treated with different concentrations of YGJDSJ or equal volume of DMEM for 24 h. YGJDSJ treated Bel-7402 cells were collected, stained with Annexin V-FITC and PI as recommended by the manufacturer, and detected in a FACScalibour flow cytometer (Becton Dickinson).
4
Transfection of miR-145-5p in Cultured Cells
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After 72 hours, cultured cells were transfected (50
nM final concentration) with miR-145-5p mimic
(Qiagen, Germany) using X-treme gene (Roche,
Germany). The culture medium was refreshed and
cell culture was continued for 24 hours. Treatment
with X-treme gene alone was applied as mock control.
PBMCs in some wells were transfected with Label
IT® RNAi Delivery Control (Mirus, USA) both as an
indicator of transfection efficiency and as a scrambled
siRNA (Fig .1 ). Transfection efficiency was analyzed
by FACS Calibour flow cytometer (Becton Dickinson)
and CellQuestTM Pro software.
nM final concentration) with miR-145-5p mimic
(Qiagen, Germany) using X-treme gene (Roche,
Germany). The culture medium was refreshed and
cell culture was continued for 24 hours. Treatment
with X-treme gene alone was applied as mock control.
PBMCs in some wells were transfected with Label
IT® RNAi Delivery Control (Mirus, USA) both as an
indicator of transfection efficiency and as a scrambled
siRNA (
by FACS Calibour flow cytometer (Becton Dickinson)
and CellQuestTM Pro software.
5
CD34+ Cell Differentiation Analysis
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The differentiation of CD34+ cells was investigated by flow cytometric analysis (BD FACS Calibour, USA). The cells were labeled with anti-CD71-APC and anti-glycophorin A-PE (BD Biosciences, USA) (1/10) using 1% BSA (w/v) in PBS for 20 minutes at 4°C on days 0, 7 and 15 of culture. The cells were analyzed using 2-color flow cytometry by combining PE-labelled and APC-labelled antibodies and the analysis was implemented on a BD FACS Calibour flow cytometer (BD Biosciences, USA) and FlowJo X10 (Tree Star Inc., USA) software.
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The differentiation of CD34+ cells was investigated by flow cytometric analysis (BD FACS Calibour, USA). The cells were labeled with anti-CD71-APC and anti-glycophorin A-PE (BD Biosciences, USA) (1/10) using 1% BSA (w/v) in PBS for 20 minutes at 4°C on days 0, 7 and 15 of culture. The cells were analyzed using 2-color flow cytometry by combining PE-labelled and APC-labelled antibodies and the analysis was implemented on a BD FACS Calibour flow cytometer (BD Biosciences, USA) and FlowJo X10 (Tree Star Inc., USA) software.
6
Phenotypic Analysis of hNSSCs
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Phenotypic analysis of hNSSCs was performed as described in our previous study (Vishnubalaji et al., 2012b (link)). Briefly, cells were washed twice in ice-cold PBS supplemented with 0.5% BSA and re-suspended at 106 cells per ml. Ten microliters of PE-conjugated mouse anti-human CD146, CD73, CD29 and HLA-DR, FITC-conjugated mouse anti-human CD34, CD90, CD45, CD13, CD184 and CD31, or APC-conjugated mouse anti-human CD105, CD14 and CD44 antibodies (all from BD Biosciences, except the anti-human CD105, which was purchased from R&D systems) was added to 100 μl of cell suspension (105 cells). Negative control staining was performed using a FITC, PE, or APC-conjugated mouse IgG1 isotype control antibodies, respectively. Cells were incubated for 30 min at 4 °C in the dark, then were washed with PBS to remove excess antibodies, and then were resuspended in 500 μL of PBS and were analyzed using BD FACS Calibour flow cytometer (BD Biosciences. Data were analyzed using Cell Quest Pro Software Version 3.3 software (BD Biosciences).
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