Immunofluorescence assays were performed by fixing ZIKV-infected cells to black-walled, optical bottom 96 well plates (ThermoFisher) with Cytofix/Cytoperm Solution (BD) for 20 minutes and washed with 1× Perm/Wash buffer (BD). Cells were blocked with normal mouse serum (1:10, Sigma) for 10 minutes at room temperature, and incubated with rabbit CXCR3 IgG monoclonal antibody (mAb) (1:250, ThermoFisher, 6H1L8) for detection of membrane bound and cytosolic CXCR3 for 1 hour at 37°C. Cells were washed with 1× Perm/Wash buffer and incubated with AlexaFluor594 goat anti-rabbit IgG secondary antibody (1:500, Invitrogen, A11012) at 37°C for 1 hour. Cells were washed with Perm/Wash buffer, stained with NucBlue reagent (Invitrogen) and stored in 1× PBS at 4°C prior to imaging. To detect only membrane bound CXCR3, the same procedure was used with Cytofix reagent (BD) for fixation and 1× PBS with 2% FBS for washing. Imaging was performed on a Nikon A1-R Confocal Microscope.
Cytofix reagent
Manufactured by BD
Cytofix is a reagent used in flow cytometry applications. It is a fixative solution designed to preserve the structure and morphology of cells during sample preparation. The Cytofix reagent helps maintain the integrity of cellular components, allowing for the analysis of cellular characteristics and properties.
Automatically generated - may contain errors
Lab products found in correlation
Normal mouse serum, by Merck Group (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Alexa fluor 594 goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Nucblue reagent, by Thermo Fisher Scientific (1 mentions)
Perm wash buffer, by Thermo Fisher Scientific (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cellcarrier, by PerkinElmer (1 mentions)
Anti tra1 60 vio488, by Miltenyi Biotec (1 mentions)
Perm wash reagent, by BD (1 mentions)
Operetta high content imaging system, by PerkinElmer (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Facscanto analyzer, by BD (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
4 protocols using cytofix reagent
1
Immunofluorescence Imaging of CXCR3 in ZIKV-Infected Cells
2
Evaluating Antibody Polyreactivity
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Polyreactivity was evaluated by flow cytometry and ELISA. For flow cytometry experiments, HEK293 cells were incubated with preimmune and mutated IGHV4-34 antibodies for 30 min at 4°C. After washing, anti–human IgG–BV605 was added for 30 min at 4°C and analyzed on a CANTO II flow cytometer. HEK293 cells were also permeabilized with Cytofix reagent (BD) to assess binding of antibodies to intracellular self-antigens. Polyreactivity ELISAs were performed as described previously using dsDNA and LPS (Tiller et al., 2008 (link)). For both flow cytometry and ELISA assays, established polyreactivity control antibodies were used: eiJB40, a low-positive control, and mGO53, a negative control antibody (Wardemann et al., 2003 (link)). These antibodies were produced as IgG1 as described in the IGHV4-34 antibodies section.
3
Pluripotency Marker Expression in iPSCs
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For staining, iPSCs were grown in 96-well imaging plates (CellCarrier, PerkinElmer) in E8 medium. After 3 days, cells were fixed with Cytofix reagent, followed by blocking and permeabilization with PermWash reagent (both from BD Biosciences). Then, cells were incubated with the dye-conjugated antibodies anti–TRA-1-60–Vio488 (Miltenyi Biotec, REA157, 130-106-872), anti-SSEA4–PerCP-Vio700 (Miltenyi Biotec, REA101, 130-105-053), anti-OCT3/4 (Isof. A)–APC (Miltenyi Biotec, REA338, 130-105-555), and anti-NANOG (D73G4)–PE (Cell Signaling Technology, 14955). Nuclei were stained with Hoechst 33342 (2.5 μg/ml in PBS; Invitrogen). Images were captured using an Operetta high-content imaging system (PerkinElmer).
4
Flow Cytometric Analysis of CD19 CAR-T Cells
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Frozen PBMCs of selected patients were thawed and flow cytometry (FC) was performed as recently described (17 (link)). In brief, CD19 CAR-expressing T cells were determined using the CD19 CAR detection reagent Biotin (cat. no. 130115965; Miltenyi Biotec GmbH) following the manufacturer's protocol. In brief, PBMCs were washed with FC buffer [PBS (Gibco, Thermo Fisher Scientific)] containing 2% fetal bovine serum (MilliporeSigma) and resuspended in 100 µl FC buffer. Cells were stained with CD19 CAR Detection Reagent Biotin for 10-15 min at RT, washed twice with FC buffer and stained with anti-CD45 Vioblue, anti-CD3 FITC and anti-biotin phycoerythrin (cat. no. 130110951; Miltenyi Biotec GmbH) for 10 min at RT. After washing with FC buffer, cells were resuspended in 500 µl FC buffer supplemented with Cytofix reagent (BD Biosciences) and subsequently analyzed on a FACS Canto Analyzer (BD Biosciences). At least 125,000 cells were analyzed in the lymphocyte gate to ensure high accuracy. Dead cells were excluded using a fixable viability dye (eFluor 506; Ebioscience; Thermo Fischer Scientific, Inc.). Results were analyzed using FlowJo software, version 10.6.2 (BD Biosciences).
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