To visualize immunological synapses, 200 μL of U87MG cells (50,000 cells/mL) were seeded onto wells of a 24-well plate and incubated at 37 oC for 12 h to allow cells to attach. 200 μL neutrophils (500,000 cells/mL) were then added onto the target U87MG cells and incubated for 6 h before fixation with 4% paraformaldehyde (in PBS). Cytoskeleton staining was then performed using an F-actin Visualization Biochem Kit (Cytoskeleton Inc.). To quantify the immunological synapses, a total of 1 × 106 CAR hPSC-neutrophils or CAR hPSC-neutrophils@R-SiO2-TPZ labeled with anti-CD45-APC (BD Biosciences) were incubated with 2 × 105 targeted cells stained with Calcein-AM fluorescent dye (Invitrogen) for various time points at 37oC and humidified 5% CO2 atmosphere. After incubation, the cells were fixed and analyzed in an Accuri C6 plus cytometer (Beckton Dickinson) after washing with BSA-containing PBS−/− solution, and cells of double-positive events APC + /Calcein+ were analyzed to quantify immunological synapse formation in FlowJo software.
Calcein am fluorescent dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Calcein-AM is a fluorescent dye used in cell biology and biochemistry. It is a cell-permeant dye that can be used to measure cell viability and cytotoxicity. When it enters a live cell, the acetomethoxy (AM) group is cleaved by intracellular esterases, converting the dye into a green-fluorescent product that is retained within the cell.
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Lab products found in correlation
Synergy ht, by Agilent Technologies (2 mentions)
F actin visualization biochem kit, by Cytoskeleton (1 mentions)
Anti cd45 apc, by BD (1 mentions)
Accuri c6 plus cytometer, by BD (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Laminin, by BD (1 mentions)
Collagen 1, by Merck Group (1 mentions)
Recombinant vcam 1, by R&D Systems (1 mentions)
Sylgard 184 polydimethylsiloxane, by Dow (1 mentions)
Ix71 inverted fluorescence microscope, by Olympus (1 mentions)
Ethidiumhomodimer 2, by Thermo Fisher Scientific (1 mentions)
Axiovert 200m microscope, by Yokogawa (1 mentions)
Plan neofluar 5x objective, by Zeiss (1 mentions)
Synergy ht fluorescence plate reader, by Agilent Technologies (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid disodium salt, by Merck Group (1 mentions)
Dextran sulfate sodium salt, by Merck Group (1 mentions)
Mtt cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Poly allylamine hydrochloride pah, by Merck Group (1 mentions)
10 protocols using calcein am fluorescent dye
1
Quantifying Immunological Synapse Formation
2
Adhesion Assay for Integrin-Mediated Cell Attachment
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We coated 96-well microplates with fibronectin (10 μg/ml in PBS; Millipore, Billerica, MA), collagen I (10 μg/ml in PBS; Sigma-Aldrich), laminin (10 μg/ml in PBS; BD Biosciences, Franklin Lakes, NJ), or 10% FBS as control. For the VCAM-1 adhesion assay, 10 μg/ml recombinant VCAM-1 (R&D) was used to coat wells. Cells were labeled for 20 min with 2 μM calcein AM fluorescent dye (Invitrogen) in Hank's buffered salt solution (HBSS). After two washes with HBSS, the cells were plated at 100,000 cells/well and incubated at 37°C for 2 h. The microplate was washed to remove nonadherent cells, and the remaining adherent cells were measured using a fluorescence plate reader with excitation wavelength of 488 nm and emission detected at 512 nm. Fluorescence data were then normalized to the mean fluorescence obtained for control cells. To measure α4β1-specific adhesion, cells were either treated with dimethyl sulfoxide or blocked with the monovalent peptide LDV (1 μM), which was a generous gift from Larry Sklar and Tione Buranda (University of New Mexico).
3
Protocatechuic Acid-Mediated Platelet Activation
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Protocatechuic acid (content ≥97.0%, Aladdin), Sylgard 184 polydimethylsiloxane (Dow Corning, USA), calcein Am fluorescent dye (Invitrogen), CD42b antibody (Biyuntian Biotechnology Company), dimethyl sulfoxide (Biyuntian Biotechnology Company), acetylsalicylic acid (Biyuntian Biotechnology Company), tirofiban (Broad Pharmaceutical (China) Co., Ltd.), ristocetin (HYPHEN), ADP (Tali Kangxin), collagen (Tali Kangxin), kaolin activator (Haemoscope, USA), sodium citrate venous blood vacuum tube (Shandong Weigao), thromboelastometer (Haemoscope, USA), RSP01-CS two-way push-pull precision injection pump (Jiashan Ruichuang), IX71 inverted fluorescence microscope (Olympus), and plasma cleaner (PDG-32G-2) were used in this study.
4
Live/Dead Cell Imaging in 3D Cultures
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3D cell cultures were double-stained with calcein AM fluorescent dye (Molecular Probes, Eugene, OR, USA) and ethidium homodimer-2 (Invitrogen, Carlsbad, CA, USA). Confocal images were acquired with a Zeiss Axiovert-200M microscope, equipped with Yokogawa CSU22 spinning disc confocal unit using Zeiss Plan-Neofluar 5× objective. Intensity projections were created with SlideBook (Intelligent Imaging Innovations Inc., Denver, CO, USA). Background noise was removed by normalization, using either SlideBook or ImageJ (NIH, Bethesda, MD, USA) programs.
5
Multicellular Structure Analysis Protocol
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Multicellular structures were double-stained with Calcein-AM fluorescent dye (Molecular Probes, C1430) and Ethidium homodimer-1 (Molecular Probes, E1169). 3D confocal images were acquired using Zeiss Plan-Neofluar 5x objective. Maximum intensity projections were created and background noise was removed by normalization with SlideBook. Images were analyzed using VTT’s in-house developed proprietary software AMIDA, software specifically designed for multicellular structure segmentation and measurement of biologically important morphological features such as size, roundness and cellular invasiveness [65 (link)]. All statistical analyses and plotting of numerical data (post-image analysis) were performed using R, an open source programming language and software environment for statistical computing and graphics (http://cran.r-project.org ).
6
ADAM15 Regulates Monocyte Adhesion
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The adhesion of THP-1 cells to control siRNA-treated or ADAM15 siRNA-treated HUVECs grown to confluence in 96-well plates was examined. HUVECs were serum-starved overnight. The next day, the cells were treated with TNF-α (10 ng/mL) for 24 h. THP-1 cells were collected and labeled with Calcein AM fluorescent dye (Life Technologies, 5 μM) for 30 min. After being washed twice, 1 × 105 THP-1 cells were added to each well and incubated for 30 min at room temperature. Nonadherent cells were washed away, and the fluorescence was measured using a Synergy HT fluorescence plate reader (BioTek Instruments, Winuski, VT, USA).
7
THP-1 Cell Adhesion Assay on RA FLSs
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The adhesion of THP-1 cells to control siRNA-treated or ADAM-17 siRNA-treated RA FLSs grown to confluence in 96-well plates was examined. RA FLSs were serum-starved overnight. The next day, the cells were treated with TNF-α (10 ng/ml) for 24 h. THP-1 cells were collected and labeled with calcein AM fluorescent dye (Life Technologies, 5 μM) for 30 min. After being washed twice, 1 × 105 THP-1 cells were added to each well and incubated for 30 min at room temperature. Nonadherent cells were washed away, and the fluorescence was measured using a Synergy HT fluorescence plate reader (BioTek Instruments, Winooski, VT, USA).
8
Adhesion Assay of THP-1 Cells to RA Fibroblasts
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Adhesion of THP-1 cells to nontreated, control siRNA or fut1 siRNA treated RA synovial fibroblasts grown to confluence in 96-well plates was examined [25 (link)]. RA synovial fibroblasts were serum-starved overnight. The next day, cells were treated with TNF-α (25 ng/ml) for 24 hours. THP-1 cells were collected and labeled with 5 μM Calcein AM fluorescent dye (Life Technologies) for 30 minutes. After washing twice, 1 × 105 THP-1 cells were added to each well and incubated for 30 minutes at room temperature. Nonadherent cells were washed off and fluorescence was measured using a Synergy HT fluorescence plate reader (BioTek Instruments, Winooski, VT).
9
Quantifying Statin-Induced Cell Viability
10
Robust Multilayered Polyelectrolyte Capsules
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Poly(4-styrene sulfonate) sodium salt (PSS, Mw = 70 kDa), poly(allylamine hydrochloride) (PAH, Mw = 17.5 kDa), dextran sulfate sodium salt (DS, Mw = 40 kDa), ethylenediaminetetraacetic acid disodium salt, calcium chloride, sodium carbonate, and Rhodamine B were purchased from Sigma-Aldrich (Taufkirchen, Germany). Dimethyl sulfoxide was purchased from Merck (Darmstadt, Germany). Cyanine 7 NHS ester (Cy7) was obtained from Lumiprobe (Moscow, Russia). Polyvinyl alcohol (PVA) powder (Mw = 72,000 g/mol with a degree of hydrolysis of 85–89%) was supplied by AppliChem GmbH (Darmstadt, Germany). All the chemicals were used without further purification. Deionized water produced using a Milli-Q Plus 185 water treatment system (Merck Millipore, Darmstadt, Germany) was used to prepare all solutions. Phosphate-buffered saline (PBS), Calcein AM fluorescent dye, and the MTT cell proliferation assay kit were purchased from Thermo Fisher Scientific (Eugene, OR, USA).
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