To analyze the binding of Spike protein to CTLs, 0.15 × 106 freshly purified CD8+ T cells (day 0) and CTLs (day 7) were pre-treated with Spike W as described in “CTL pre-treatments”. Cells were then labelled with anti-Spike human J08 mAb for 30 min on ice. After washing with cold PBS, samples were incubated for 30 min on ice with Alexa Fluor 488- anti-human (#A11013, ThermoFisher Scientific) secondary antibody and then analyzed by confocal microscopy. Alternatively, 0.15 × 106 CTLs (day 7) were incubated with MiniV or control liposomes as described in “CTL pre-treatments” and analyzed by confocal microscopy.
Alexa fluor488 anti human
Manufactured by Thermo Fisher Scientific
Alexa Fluor488 anti-human is a fluorescently labeled antibody that binds to human proteins. It is designed for use in various immunoassay and flow cytometry applications to detect and quantify human target proteins.
Automatically generated - may contain errors
4 protocols using alexa fluor488 anti human
1
Spike Protein Binding to CTLs
2
Immunofluorescence Staining of Centromeres
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NIH 3T3 and HEK293T cells were seeded in 35mm glass-bottom imaging dishes (MatTEK). Cells were then pre-permeabilized with 0.25% of Triton X-100 for 60 s and fixed for 10 min in 100 mM HEPES (pH 7; titrated with KOH), 50 mM EGTA (pH 7; titrated with KOH), 2% formaldehyde (methanol free) and 0.2% Triton X-100. Primary antibody was diluted in PBS, 3% BSA and 0.1% Triton X-100 and the cells were incubated for 1.5h at room temperature (ACA centromere CREST autoantibody, FZ90C-CS1058, Europa Bioproducts; 1:500). Alexa Fluor488 anti-human (Thermo Fisher; 1:500) was used as a secondary antibody and DNA was counterstained with 5 mg/ml Hoechst 33342 (Molecular Probes).
3
Spike Protein Binding to CTLs
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To analyze the binding of Spike protein to CTLs, 0.15 × 106 freshly purified CD8+ T cells (day 0) and CTLs (day 7) were pretreated with Spike W, as described in “CTL pretreatments.” Cells were then labeled with anti-Spike human J08 mAb for 30 min on ice. After washing with cold PBS, samples were incubated for 30 min on ice with Alexa Fluor 488–anti-human (#A11013; Thermo Fisher Scientific) secondary Ab and then analyzed by confocal microscopy. Alternatively, 0.15 × 106 CTLs (day 7) were incubated with MiniV or control liposomes as described in “CTL pretreatments” and analyzed by confocal microscopy.
4
Visualizing Kinetochore-Microtubule Dynamics
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Before fixation, kinetochore-bound microtubules were selectively depolymerised by exposing the oocytes to 4 °C for 14 min. Following the cold-treatment, the dish was removed from ice and oocytes were permeabilised by a brief 10 s exposure to 0.25% Triton X-100. Oocytes were then fixed for 30 min at 37 °C in 100 mM HEPES (pH 7; titrated with KOH), 50 mM EGTA (pH 7; titrated with KOH), 2% formaldehyde (methanol-free), and 0.2% Triton X-100. Thereafter, oocytes were extracted overnight at 4 °C in PBS supplemented with 0.1% Triton X-100. All antibody incubations were performed in PBS, 3% bovine serum albumin, and 0.1% Triton X-100, either overnight at 4 °C (primary antibodies) or for 3 h at room temperature (secondary antibodies). Primary antibodies used were human ACA centromere CREST autoantibody (FZ90C-CS1058, Europa Bioproducts; 1:500) and rat anti-α-tubulin (MCA78G, Serotec; 1:1000). As secondary antibodies, Alexa Fluor488 anti-human and Alexa Fluor647 anti-rat (Thermo Fisher; 1:400) were used. DNA was stained with 5 mg/ml Hoechst 33342 (Molecular Probes).
Fixed oocytes were imaged using the AiryScan module on Zeiss LSM800 microscope equipped with 40× C-Apochromat 1.2 NA water-immersion objectives and processed post-acquisition using ZEN2. Images were acquired at a spatial resolution of 0.19–0.30 μm optical sections, covering the entire spindle.
Fixed oocytes were imaged using the AiryScan module on Zeiss LSM800 microscope equipped with 40× C-Apochromat 1.2 NA water-immersion objectives and processed post-acquisition using ZEN2. Images were acquired at a spatial resolution of 0.19–0.30 μm optical sections, covering the entire spindle.
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