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).
Alexa fluor 647
Manufactured by Bio-Rad
Sourced in United States
Alexa Fluor 647 is a highly fluorescent dye that absorbs and emits light at specific wavelengths. It is commonly used in various biological and biomedical applications, such as fluorescent labeling and detection.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti bovine cd4 fitc, by Bio-Rad (1 mentions)
Mouse anti bovine cd21 rpe, by Bio-Rad (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Facscalibur, by BD (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
Alexa fluor 555 goat anti mouse igg1 antibody, by Thermo Fisher Scientific (1 mentions)
Cellquest version 3, by BD (1 mentions)
Facs scan cytometer, by BD (1 mentions)
Cd16 cd32, by BioLegend (1 mentions)
Cd4 apc, by BioLegend (1 mentions)
Cd11c pe cy7, by BioLegend (1 mentions)
Cd3 pe cy7, by BioLegend (1 mentions)
Collagenase 2, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
F4 80, by Bio-Rad (1 mentions)
Multiplex assay, by Bio-Rad (1 mentions)
Elisas, by Thermo Fisher Scientific (1 mentions)
9 protocols using alexa fluor 647
1
Immunological Profiling of Sheep
2
Quantifying T and B Cells by Flow Cytometry
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Antibody staining of cells and flow cytometric quantification of T and B cells were described in detail by Liermann et al. (28 (link)). Briefly, cells were stained by monoclonal antibodies for cluster of differentiation (CD)2 (T cells) (mouse anti-bovine CD2: FITC; Bio-Rad Laboratories, Hercules, USA), CD4 (T helper cells) (mouse anti-bovine CD4: FITC; Bio-Rad), CD8 (cytotoxic cells) (mouse anti-bovine CD8: Alexa Fluor® 647; Bio-Rad), and CD21 (B cells) (mouse anti-bovine CD21: RPE, Bio-Rad) or the corresponding isotype controls (mouse IgG1 negative control: FITC; mouse IgG2a negative control: FITC; mouse IgG2a negative control: Alexa Fluor® 647; mouse IgG2b negative control: RPE; Bio-Rad). A flow cytometer Type GalliosTM (Beckman Coulter GmbH, Krefeld Germany) was used.
3
HLA-A2 Positive Donor Recruitment
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This study was reviewed and approved by the ethic committee of Hannover Medical School, approval number 2315-2014. All healthy donors (n = 9) were recruited in Hannover Medical School for a previous study, in which their HEV seroprevalence were tested negative (11 (link)). HLA phenotyping on all donors was done by antibody staining (mouse anti-human HLA-A2, clone BB7.2, Alexa-Fluor 647; Bio-Rad Laboratories, USA); all individuals were HLA-A2 positive. Written informed consents for participating in this research study and blood draws were collected from all individuals.
4
Adenoviral FasL Expression Assay
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Hepa1–6 cells were transduced with 5 TU/cell Ad-cFasL or Ad-gFasL, and after various periods, FasL expressed on the cell surface was analyzed. BAECs were transduced with 10–50 TU/cell Ad-cFasL or Ad-gFasL, and after 48 h, the cells were harvested and FasL expression was determined. FasL was detected on the surface of the transduced cells with anti-FasL antibodies (MLF4 clone) labeled with Alexa Fluor 647 (#MCA 2896A647 from BioRad, CA, USA), using the CytoFlex (Beckman Coulter, Indianapolis, IN, USA).
5
Analyzing T Cell Activation via Strep-Tactin
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CHO mock or CHO ligand anchor HLA-A*02 cells (2 × 105) preincubated with trivalent Strep-Tactin-SpyCatcher or buffer alone were mixed with untransduced Jurkat NFκB eGFP cells or Jurkat NFκB eGFP 1G4 TCRα/β-Twin-Strep-tag cells (1 × 105) in DMEM 5% FBS, 100 U mL−1 penicillin/streptomycin, 2 μg mL−1 avidin. Alternatively, CHO mock or CHO ligand anchor HLA-A*02 cells (2 × 105) were mixed with untransduced Jurkat NFκB eGFP cells or Jurkat NFκB eGFP 1G4 TCRα/β-Twin-Strep-tag cells (1 × 105), and NY-ESO-1 9V peptide in DMEM 5% FBS, 100 U mL−1 penicillin/streptomycin, 2 μg mL−1 avidin. Cells were incubated in a 37°C 10% CO2 incubator for 20 hours. Cells were harvested, incubated with anti-CD45 antibody Alexa Fluor 647 (F10-89-4; Bio-Rad Laboratories), and analysed by flow cytometry (BD FACSCalibur, 640-nm laser, FL4–661/16 band-pass filter; 488-nm laser, FL1–530/30 band-pass filter, BD Biosciences).
6
Cardiac Macrophage Phenotyping by Flow Cytometry
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For the flow cytometry analysis, heart tissue was digested by type II collagenase (Sigma) at 37 °C for 40 to 50 min while rocking. Next, 10% FBS was added to terminate enzyme activity. The cell suspension was filtered through a 75 µm cell strainer and centrifuged at 1500 rpm for 10 min. For the in vitro experiments, macrophages from different treatment groups were collected using sterile PBS. Cells from the heart tissue or the in vitro experiment were incubated with antibodies against CD68 (Alexa Fluor 647, AbD Serotec), CD11c (FITC, AbD Serotec) and CD163 (RPE, AbD Serotec) at room temperature and kept in the dark for 20 min. Cells were then washed with PBS and analyzed using a FACSCalibur flow cytometer.
7
Multiparametric Immunofluorescence Staining
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Slides underwent fluorescent staining as described previously [2 (link),28 (link)]. In short, cells were incubated with an antibody mix consisting of conjugated mouse anti-human CD45 Alexa Fluor® 647 (clone: F10-89-4, MCA87A647, AbD Serotec, Raleigh, NC, USA), a cocktail of mouse IgG1/Ig2a anti-human cytokeratins (CK) 1, 4, 5, 6, 8, 10, 13, 18, and 19 (clones: C-11, PCK-26, CY-90, KS-1A3, M20, A53-B/A2, C2562, Sigma, St. Louis, MO, USA), mouse IgG1 anti-human CK 19 (clone: RCK108, GA61561-2, Dako, Carpinteria, CA, USA), and rabbit IgG anti-human vimentin (Vim) Alexa Fluor® 488 conjugated (clone: D21H3, 9854BC, Cell Signaling, Danvers, MA, USA) for 2 h. Slides were then incubated with Alexa Fluor® 555 goat anti-mouse IgG1 antibody (A21127, Invitrogen, Carlsbad, CA, USA) and counter-stained with 4′,6-diamidino-2-phenylindole (DAPI; D1306, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Slides were finally mounted with a glycerol-based aqueous mounting media to enable future coverslip removal for downstream genomic and proteomic analyses without disrupting cell integrity.
8
Single-Cell Isolation of Murine Skin Cells
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Mice were euthanized, and single-cell suspensions were obtained from the full-thickness tail skin distal to the surgical margin. The tissue was minced and incubated in 5 mg/mL collagenase II and 0.05 mg/mL DNase (both from Sigma) in RPMI 1640 medium (Gibco, Thermo Fischer) for 30 minutes at 37 C. Single-cell suspensions were filtered through 70-and 40-mm cell strainers and resuspended in FACS buffer (2% fetal bovine serum, 2 mmol/L EDTA). Cells were incubated with CD16/CD32 (1:100; Biolegend, San Diego, CA) to block endogenous Fc receptors and live/ dead staining was performed using Fixable Aqua Zombie (1:300; Invitrogen). Cell populations were analyzed using multicolor surface markers for myeloid and lymphoid populations. Myeloid panel: CD45 (Pacific blue, 1:400), Ly6G (fluorescein isothiocyanate, 1:250), Ly6C (PE, 1:125), CD11b (PerCPC5.5, 1:400), CD11c (PECy7, 1:400) (all from Biolegend), and F4/80 (Alexa Fluor 647; AbD Serotec, Oxford, UK; 1:400). Lymphocyte panel: CD45 (Pacific blue, 1:400), CD3 (PECy7, 1:200), CD4 (APC, 1:200), CD8 (fluorescein isothiocyanate, 1:400) (all from Biolegend).
9
Immunofluorescence Staining of Colon Macrophages and Neutrophils
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The distal colon was fixed with 4% paraformaldehyde. Frozen sections (5 m thick) were prepared for immunofluorescen ce staining as previously described (Azuma et al., 2008) , with minor modifications. Macrophages were detected using rat anti-mouse F4/80 monoclonal antibod ies conjugated to Alexa Fluor 647 (CI:A3-1; AbD Serotec). Neutrophils were detected using rat anti -mouse After 48 h of culture, cytokine concentrations were determined in the culture supernatants using enzyme-linked immunosorbent assays ( ELISAs;
eBioscience, San Diego, CA , USA) and multiplex assays (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions .
eBioscience, San Diego, CA , USA) and multiplex assays (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions .
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