Staphylococcal log cultures were labeled with PBS 50 μM 5-chloromethylfluorescein diacetate (CMFDA) (Life Technologies, Carlsbad, CA, USA) for 1.5 h in a 37 °C waterbath. The labeling was quenched in 2% bovine serum albumin (BSA) PBS (PBSB). The staphylococci were washed with cold PBSB and stained on ice with PBSB containing 10 μg/mL MHCII tetramer (NIH, Bethesda, MD, USA) for 45 min. Freshly isolated splenocytes were blocked with 2% ICS PBS (PBSI) and mixed with 100 μL CMFDA-labeled staphylococci on ice for 25 min. The stainings were acquired in an LSRFortessa (BD, San Jose, CA, USA). For sorting, the staphylococci were washed with cold PBSB, mixed on ice with PBSB containing GFP for 45 min, and run through a FACSAria (BD, San Jose, CA, USA).
5 chloromethylfluorescein diacetate cmfda
Manufactured by Thermo Fisher Scientific
Sourced in United States
5-chloromethylfluorescein diacetate (CMFDA) is a fluorogenic compound that can be used for cellular labeling and tracking. It is cell-permeant and, upon hydrolysis by intracellular esterases, produces a fluorescent product that is retained within the cell.
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Lab products found in correlation
60 mm culture dishes, by BD (3 mentions)
Laminin, by Merck Group (2 mentions)
Vitronectin, by BD (2 mentions)
Facstar flow cytometer, by BD (2 mentions)
Cmfda, by BD (2 mentions)
Milli q plus water, by Merck Group (2 mentions)
Jc1 mitoscreen assay, by BD (2 mentions)
Single laser facscalibur, by BD (2 mentions)
Dichloro dihydro fluorescein diacetate dcfh da, by Merck Group (2 mentions)
Facscalibur, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Dioc2 3, by Thermo Fisher Scientific (2 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
3 3 diethyloxacarbocyanine iodide dioc2 3, by Thermo Fisher Scientific (2 mentions)
Facsaria, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Dms300 digital microscope, by Leica (1 mentions)
Metamorph automation software, by Molecular Devices (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
7 aminoactinomycin d 7 aad, by Thermo Fisher Scientific (1 mentions)
29 protocols using 5 chloromethylfluorescein diacetate cmfda
1
Staphylococcal Immune Cell Interactions
2
Flow Cytometry Analysis of T-Cell-MSC Conjugation
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T cells (1 × 106 cells/ml) were labeled with 0.5 μM 5-chloromethylfluorescein diacetate (CMFDA) (Life Technologies) and MSCs with 5 μM 5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (Thermo Fisher Scientific) in serum-free medium for 10 min at 37 °C. After staining, cells were washed twice in culture medium having 10% FBS. MSCs (1 × 105) and T cells (1 × 106) were mixed in a 12 × 75-mm polystyrene tube (BD Biosciences) and centrifuged at 1000 rpm for 1 min, and pellets were incubated at 37 °C for 2 h. Cell mixtures were then gently suspended and analyzed by flow cytometry. The conjugation ratio was calculated as the portion of CMFDA/CMTPX double-positive events.
3
LCZ636 Inhibits Monocyte-Endothelial Adhesion
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U937 monocytes were labeled with 15 μM 5-chloromethylfluorescein diacetate (CMFDA) (Life Technologies) for 45 minutes at 37°C followed by washing twice with the medium. For endothelial cell-monocyte adhesion, HUVECs were stimulated with 1 μg/mL LPS in the presence or absence of 10 and 20 μM LCZ636 and incubated with CMFDA-labeled U937 monocytes for 30 minutes at 37°C, followed by washing twice with warm PBS. The number of adherent U937 cells per visible field was determined by recording a video using the Leica DMS300 digital microscope (Leica, Shanghai), Images were recorded and the attached cells were quantified from 10 frames in the off-line analysis using Metamorph automation software (Molecular Devices, USA).
4
Murine Cell Culture and Staining
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Murine Yac-1, B16-F10, EL4 cells as well as ovalbumin (OVA)257–264 peptide-expressing EL4-OVA8 cells and GFP-expressing B16-GFP and EL4-OVA8-GFP cells were cultured in RPMI-1640 medium (Gibco, MA, USA) supplemented with 10% FCS (Gibco, MA USA). Recombinant mouse cytokines, including granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-2, IL-4, IL-6, IL-7, and IL-15, were obtained from PeproTech (NJ, USA). The fluorescent dyes used for cell staining were 5-chloromethylfluorescein diacetate (CMFDA), Hoechst 33,342, and 7-aminoactinomycin D (7-AAD; Life Technologies, MA, USA).
5
Adhesion Assay for Endothelial Cells
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An adhesion assay was performed as previously described [43 (link)]. bEnd.3 cells, grown in 24-well plates, were treated with LPS at 37 °C for 24 h after pretreatment with chrysin for 30 min and then washed twice with PBS. U937 cells were labeled for 30 min at 37 °C with 2 μM 5-chloromethylfluorescein diacetate (CMFDA, Molecular Probes), washed twice with PBS, and suspended in growth medium. Then, 2.5 × 105 labeled cells were added to the bEnd.3 monolayer at a final volume of 500 μL and incubated in a CO2 incubator for 2 h at 37 °C on a block light. Non-adherent cells were removed from the plate by gentle washing with PBS, and the number of adherent cells was determined by measuring the fluorescence intensity under a fluorescence microscope (Carl Zeiss, Germany).
6
Quantifying Cancer Cell Invasion and Adhesion
7
Cellular GSH Levels Analysis
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Cellular GSH levels were analyzed using 5-chloromethylfluorescein diacetate (CMFDA; Ex/Em = 522 nm/595 nm; Invitrogen Molecular Probes) as previously described6 (link),37 (link). In brief, 1 × 106 cells in 60-mm culture dishes (BD Falcon) were pretreated with 2 mM NAC or 10 μM BSO for 1 h and then treated with PG at an indicated concentration (100–1600 μM) for 24 h or other indicated time points. Cells were then washed with PBS and incubated with 5 µM CMFDA at 37 °C for 30 min. CMF fluorescence intensity was determined using a FAC Star flow cytometer (Becton Dickinson). CMF-negative (−) staining cells indicate depletion of GSH in cells. The mean CMF levels in cells, except for CMF-negative (GSH-depleted) cells, were expressed as a percentage of those of control cells.
8
Quantifying Cellular Glutathione Levels
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Cellular GSH levels were evaluated using 5-chloromethylfluorescein diacetate (CMFDA, Ex/Em = 522 nm/595 nm; Invitrogen Molecular Probes), as previously described [16 (link),34 (link)]. In brief, 1 × 106 cells in 60 mm culture dishes (BD Falcon) were pretreated with each caspase inhibitor (15 μM) for 1 h and then treated with 800 μM PG for 1 or 24 h. Cells were then washed with PBS and incubated with 5 µM CMFDA at 37 °C for 30 min. The mean CMF fluorescence intensity was determined using a FACStar flow cytometer (BD Sciences). CMF-negative cells indicated the depletion of GSH content in lung cancer cells. Mean CMF levels in cells, except for CMF-negative (GSH-depleted) cells, were expressed as percentages compared with control cells.
9
Quantifying Cancer Cell Invasion and Adhesion
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Invasion and migration were measured using transwell chambers as previously described.25 (link) Briefly, 4×105 cells were added to the upper chamber and allowed to migrate for 12 h or invade into collagen-coated (15μg) membranes for 24 h at 37°C. The number of migrated and invaded cells was counted in five (200×) random fields of view. Cellular adhesion was assayed in 96-well plates pre-coated with extracellular matrix proteins (5mg/ml). Collagen I (rat tail), fibronectin, and vitronectin were purchased from BD Biosciences (San Jose, CA), laminin was obtained from Millipore (Billerica, MA). Fluorescently labeled (10 mM 5- Chloromethylfluorescein Diacetate (CMFDA, Molecular Probes) cancer cells (5×104 cells/well) were allowed to adhere for 1 h. The number of adherent cells was determined with using a standard curve and was expressed in reference to Poly-D-Lysine coated wells.
10
Investigating Macrophage ERK5 Deficiency in Atherosclerosis
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