GeneMorph II Random Mutagenesis Kit (Agilent) was used to introduce random mutations into the DNA sequence of AYC08, the nanobody used for determining the previous crystal structure23 (link). The mutation rate was estimated to be ~6 bp/1 kb. The pool of mutated DNA sequences was co-electroporated with a linearized yeast display vector, pYDS649HM47 (link), into Saccharomyces cerevisiae (strain: BJ5465). The display of the nanobody mutants on the yeast cell surface was induced by galactose. It was estimated that the sequence diversity of the mutant library was about 108. The induced cells were stained with an anti-HA antibody labeled with Alexa647 (BioLegend) and SecA-OAIns-sfGFP/SecYE23 (link). The stained cells were sorted and analyzed by flow cytometry. Nanobodies with the highest SecY affinity were enriched after several rounds of sorting. Several mutations were identified and sequenced.
Alexa 647
Manufactured by BioLegend
Sourced in United States
Alexa 647 is a fluorescent dye used in flow cytometry and other fluorescence-based applications. It has an excitation maximum at 650 nm and an emission maximum at 668 nm, making it suitable for detection in the far-red region of the spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Percp cy5, by BioLegend (3 mentions)
Pe cy7, by BioLegend (3 mentions)
Fixation buffer, by Thermo Fisher Scientific (2 mentions)
Bv421, by BioLegend (2 mentions)
Annexin 5 binding buffer, by BD (2 mentions)
Fc block, by BD (2 mentions)
Genemorph 2 random mutagenesis kit, by Agilent Technologies (1 mentions)
Albumin from chicken egg white, by Merck Group (1 mentions)
Alexa fluor 488 phalloidin, by Cell Signaling Technology (1 mentions)
Alexa fluor 647 phalloidin, by Cell Signaling Technology (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions)
Biotin, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Sa cy3, by Jackson ImmunoResearch (1 mentions)
Alexa 488, by BD (1 mentions)
Percp, by Thermo Fisher Scientific (1 mentions)
Navios, by Beckman Coulter (1 mentions)
11 protocols using alexa 647
1
Directed Evolution of Nanobody Variants
2
Proliferative Assessment of ESMG Outgrowths
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Outgrowths from the minced ESMG fragments were identified by their conspicuous protrusion over time (appearing between days 3 and 5), and their proliferative status was assessed by adding 10 μmol/L EdU to the media for 4 hours on day 7 after plating. Whole-mount labeling using the method described by Wang et al,23 (link) in combination with the aforementioned EdU Imaging Kit, was used to detect the incorporated EdU.
Whole-mount in situ labeling of spheroids was accomplished in a similar manner using CK7 at 1:1000 (Dako) and P63 at 1:1000 (Cell Signaling, Danvers, MA) primary antibodies, and anti-mouse Alexa 555 1:500 and anti-rabbit Alexa 647 at 1:500 (Biolegend, San Diego, CA) secondary antibodies, respectively. EdU (10 μmol/L) was added to live spheroid media overnight before fixation for whole-mount imaging. EdU+ cells were identified using the aforementioned EdU imaging kit. Images were acquired on an EVOS microscope and fluorescent image merging was performed using ImageJ software. In conjunction with immunofluorescence, phenotype was identified based on a solid or hollow morphology.
Whole-mount in situ labeling of spheroids was accomplished in a similar manner using CK7 at 1:1000 (Dako) and P63 at 1:1000 (Cell Signaling, Danvers, MA) primary antibodies, and anti-mouse Alexa 555 1:500 and anti-rabbit Alexa 647 at 1:500 (Biolegend, San Diego, CA) secondary antibodies, respectively. EdU (10 μmol/L) was added to live spheroid media overnight before fixation for whole-mount imaging. EdU+ cells were identified using the aforementioned EdU imaging kit. Images were acquired on an EVOS microscope and fluorescent image merging was performed using ImageJ software. In conjunction with immunofluorescence, phenotype was identified based on a solid or hollow morphology.
3
Visualizing Platelet Actin Cytoskeleton
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Platelets were diluted to a final concentration of 2 × 104 cells/mL in cell culture medium (DMEM, Sigma-Aldrich, Vienna, Austria), pipetted onto a glass slide and incubated for 15 min. Non-adhered cells were washed away with PBS (phosphate-buffered saline). For DIC microscopy, cells were fixed using 4% paraformaldehyde in distilled water and imaged. The actin cytoskeleton was visualized using Alexa Fluor 647 phalloidin and Alexa Fluor 488 phalloidin (Cell Signaling Technology, Leiden, The Netherlands), respectively. Platelet actin staining was performed in Cytoskeleton buffer with sucrose (CBS) containing 10 mM MES pH 6.1, 138 mM KCl, 3 mM MgCl2, 2 mM EGTA, and 0.32 M sucrose according to a protocol of Louise Cramer (MRC Laboratory for Molecular Cell Biology, UCL, London, UK) [24 (link)]. Briefly, platelets were fixed using 4% paraformaldehyde in CBS for 20 min at room temperature, permeabilized in 0.5% Triton X-100 with CBS, blocked in 10% albumin from chicken egg white (Sigma-Aldrich, Vienna, Austria) and stained for 20 min with 66 nM fluorophore conjugated to phalloidin. For CD62p labeling, fixed cells were stained with 1 µg/mL anti-CD62p antibody marked with Alexa 647 (BioLegend, San Diego, CA, USA) for 10 min.
4
Multicolor Flow Cytometry Analysis of Immune Cells
5
Multiparametric FACS Analysis of MSCs
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RAW 264.7 cells, MSCs, and MF-MSCs were detached using a cell scraper and Trypsin-EDTA and then made into single-cell suspensions. Cells were stained with antibodies against CD90 and F4/80 which are conjugated with FITC and Alexa 647 (Biolegend, San Diego, CA, USA), respectively. Then cells were analyzed by FACS cantoII (BD Bioscience, San Jose, CA, USA).
6
Skin Immunofluorescence Staining
7
Determination of Cellular Toxicity
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AnnexinV/PI (propidium iodide) and Ki67 stainings for determination of toxicity were performed 1 day after addition of the peptides to 105 NIH3T3 cells/well. On the second day, cells were detached using cell dissociation buffer and resuspended in 50 µl of AV/PI binding buffer [10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2]. AV (12.5 µg/µl) was added (Alexa 647, Biolegend, #640912) for 20 minutes at room temperature in the dark, and samples were measured immediately after adding PI (1 µg/ml; Biolegend, #421301) using LSR-2 (BD Biosciences, Heidelberg, Germany). Ki67 was stained using anti-mouse Ki67-PE antibody (Biolegend, 652404, 1:50).
8
Characterizing Small Bowel-Homing T Cells
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To identify small bowel homing T lymphocytes freshly isolated PBMC (105) were labeled with previously determined optimal concentrations of monoclonal antibodies directed against human CD3 (APC; eBioscience), CD4 (PerCp; eBioscience), CD49d (α4-Integrin) (FITC; BioLegend), β7-Integrin (PE; BioLegend) and CCR9 (Alexa647; BioLegend). Matching IgG1 and IgG2a conjugated antibodies served as isotype controls. Cells were incubated with the antibodies for 30 minutes on ice and washed three times in FACS buffer between each step. Nonspecific binding of antibodies was minimized by 10-minute incubation of cell suspensions with pooled human serum (10%) in fluorescence-activated cell sorting (FACS) buffer before addition of the first antibody. After washing twice, cells were re-suspended in 100 μl of 1% paraformaldehyde in PBS and analyzed utilizing a Beckman Coulter Navios. Lymphocyte populations were gated based on forward scatter/side scatter properties. A total of 3 × 104 events were routinely collected and analyzed.
9
Multiparametric Flow Cytometry of Immune Cells
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Nonadherent cells were collected, pelleted and washed with PBS prior to viability staining (Invitrogen cat no. L34957, 1:1000) for 10 minutes; cells were washed with 2% FBS in PBS for antibody staining. Cells were incubated with Fc Block (BD Biosciences cat no. 564219, 1:50) for 15 minutes prior to the addition of labeled antibodies. Cells were stained for Siglec-8 (PE-Cy7, Biolegend cat no. 347112, 1:50), CCR3 (Alexa647, Biolegend cat no. 310710, 2.5:50), CD69 (BV421, Biolegend cat no. 310930, x:50), and CD44 (PerCP-Cy5.5, Biolegend cat no. 103032, 0.5:50) for 30 minutes. Stained cells were washed with PBS and stained with Annexin V (1:100) in Annexin-V Binding Buffer (BD cat no. 556454) for 10 minutes. Cells were washed and stored in fixation buffer (eBioscience cat no. 8222-49) for 1–3 days. All staining procedures were carried out at 4°C. Prior to analysis, samples were resuspended in 2% FBS and analyzed on an LSRII or Fortessa cytometer in the Cincinnati Children’s Hospital Medical Center (CCHMC) Research Flow Cytometry Core (RFCC). Single-color compensation was calculated, and data were analyzed in FlowJo (Version 10.6.1).
10
Multiparametric Flow Cytometry of Immune Cells
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