100 µl of blood in EDTA was collected from immunized and unimmunized group of sheep at 0 days before challenge (49 DPV) and at 3DPC (52 DPV), 7 DPC (56 DPV), 14 DPC (63 DPV) and 21 DPC (70 DPV). 10 µl of conjugated antibodies, mouse anti-ovine CD4: ALEXA FLUOR®647 (Neat-1/10) dilution) and mouse anti-ovine CD8: RPE (Serotec, Immunological Excellence, USA) were mixed and incubated at room temperature for 30 min. Cells were washed with PBS, lysed with RBC lysis buffer and fixed with conjugated antibodies. Fixed cell pellets were analyzed in FACS scan cytometer (Becton Dickinson, USA). Appropriate isotype controls, mouse IgG2a negative control: RPE (for CD8) and mouse IgG2a negative control: ALEXA FLUOR®647 (for CD4) (Serotec, Immunological Excellence, USA) were used to overcome background fluorescence, if any. Stained cells were acquired in FACS scan cytometer and analyzed using software CELLQuest version 3.1 (Becton Dickinson, USA) after subtraction of the corresponding isotype control. Ten thousand events were recorded from each sample. Mean percentage variation in peripheral blood was analyzed for lymphocyte subpopulation by RPE fluorescence at (FL-2) and ALEXA FLUOR®647 fluorescence at (FL-4).
Facs scan cytometer
Manufactured by BD
Sourced in United States
The BD FACS scan cytometer is a flow cytometry instrument designed to analyze and sort cells based on their physical and fluorescent characteristics. It is capable of detecting and quantifying multiple parameters of individual cells within a sample. The core function of the BD FACS scan cytometer is to perform high-speed cell analysis and sorting.
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5 protocols using facs scan cytometer
1
Immunological Profiling of Sheep
2
Immunophenotyping of Stem Cells
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Cells were detached using 0.05% trypsin‐EDTA, recovered by centrifugation at 300 g for 5 minutes at 4°C, washed twice in PBS and re‐suspended at concentration of 105 cells per microgram antibody. Antibodies used were phycoerythrin (PE)‐ or fluorescein isothiocyanate (FITC)‐conjugated rat anti‐mouse monoclonal antibodies CD11b (clone M1/70), CD45 (clone A20), Sca‐1 (clone 13‐161.7), CD90 (clone OX‐7) purchased from BD Biosciences (Becton Dickinson, Franklin Lakes, NJ, www.bd.com ), and CD105 (clone 209701) purchased from R&D systems (R&D Systems, Minneapolis, MN, www.rndsystems.com ). Negative controls were APC‐, PE‐ or FITC‐conjugated rat anti‐mouse IgG of the same isotype as antibodies. Cells were incubated in the dark at 4°C for 30 minutes. Cells were then washed twice with PBS and fixed with 1% paraformaldehyde in phosphate‐buffered saline (PBS). Signal acquisition of fluorescence was performed using FACS‐Scan cytometer or Accuri C6 Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, www.bd.com ). FACS analyses were carried out using Weasel or CFlow‐plus software, depending on the flow cytometer.
3
Quantifying Tissue Factor Expression in Stressed Cells
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Cells were submitted to both stress for1 hr up to 8 hrs, washed, fixed with Fix and Perm® (Sigma‐Aldrich), and kept at 4°C before incubation with FITC‐conjugated (Fluorescein isothiocyanate) rabbit anti‐rat TF (dilution: 1:50; Life Science), Saint Louis, MO, USA during 30 min. in darkness. Tissue factor expression‐associated green fluorescence was quantified by flow cytometry (FACS‐scan cytometer; Becton Dickinson, San José, CA, USA) set at logarithmic gain. Around 10,000 events were recorded for each sample.
4
Apoptosis Analysis using Annexin V
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For apoptosis analysis we used the Annexin V Staining Kit (PE; 7AAD) (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer’s instructions. Subsequently, the cells were washed in phosphate-buffered saline and analyzed with a BD FACS Scan cytometer using CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA).
5
Cell Cycle Analysis by Flow Cytometry
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For cell cycle analysis, cells were trypsinized and fixed with 70% ice-cold ethanol for 30 min on 4 °C, washed twice with PBS and resuspended in 200 µL PI (from 50 µg/mL stock solution, abcam). Stained cells were analyzed on a BD FACS Scan cytometer using CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA).
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