For migration imaging, IgA-Cre/YC3.60flox mice (8–12 weeks-old) were anesthetized with a mixture of three types of anesthetic agents as described previously (19 (link)). An incision was carefully made in the abdominal wall, and the small intestine was exposed. PPs were identified by naked eyes. About 8000 sorted CD11b+IgA+ PP B cells and 30 000 CD11b−IgA+ PP B cells were labeled with CellTracker Orange CMTMR fluorescent dye (Invitrogen, USA) and then injected to a PP of an IgA-Cre/YC3.60flox transgenic mouse directly by a 25 μl syringe (Trajan Scientific and Medical, Australia). The PP with transferred cells was observed under an LSM 880 microscope (Carl Zeiss, Germany). Images were analyzed with ZEN2009 software (Carl Zeiss, Germany). After one-hour observation of the PP under a microscope, the abdominal incision was carefully closed with an ELP Skin Stapler (Akiyama Co. Ltd. Japan). Forty hours after transfer, under anesthesia, the PP with transferred cells was observed again to identify the localization of transferred CD11b+IgA+ PP B cells under an LSM 880 microscope (Carl Zeiss, Germany) and analyzed with ZEN2009 software (Carl Zeiss, Germany).
Celltracker orange cmtmr fluorescent dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
CellTracker orange CMTMR is a fluorescent dye used to label live cells. It is a cell-permeant dye that becomes fluorescent upon entering the cell and reacting with intracellular thiols, enabling the visualization and tracking of labeled cells.
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5 protocols using celltracker orange cmtmr fluorescent dye
1
In Vivo Tracking of Intestinal IgA+ B Cells
2
Tracking Inducible Germinal Center B Cells
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As described above, spleen naive B cells were cultured with pam3CSK4 (1 μg ml−1, Invivogen, USA), heat-killed E. coli (107 CFU ml−1), CpG (20 μg ml−1, GeneDsign Inc, Japan) or anti-IgM-Ab (15 μg ml−1; Jackson Immuno Research Laboratories, USA) for 3 days in vitro, separately. Then, 1 × 105 B220+ B cells were sorted from the simulated cells and subsequently seeded on 40LB feeder cells with rIL-4 (1 ng ml−1; Biolegend, USA) to induce iGB cells. Non-stimulated spleen naive B cells were seeded on 40LB feeder cells with rIL-4 (1 ng ml−1; Biolegend, USA) as a control group.
After 4-day culturing, 3 × 105 iGB cells (B220+) of naive B-40LB cells, pam3CSK4-40LB cells, E. coli-40LB, IgM-40LB and CpG-40LB cells were sorted and labeled with CellTracker Orange CMTMR fluorescent dye (Invitrogen, USA) and then separately injected intravenously to a mouse (Balb/c, 8–12 weeks old) together with 10 μg of AF488 anti-mouse MAdCAM-1 (Biolegend mAb MECA-367, USA) to identify high endothelial venules (HEVs). Under anesthesia, to stain the IgA+ cells for GC identification, 1 μg of AF647 anti-mouse IgA (Southern Biotech, USA) was directly injected to a PP of the same mouse transferred with iGB cells. The localization of transferred labeled iGB cells was observed under an LSM880 microscope (Carl Zeiss, Germany) and analyzed with ZEN2009 software (Carl Zeiss, Germany).
After 4-day culturing, 3 × 105 iGB cells (B220+) of naive B-40LB cells, pam3CSK4-40LB cells, E. coli-40LB, IgM-40LB and CpG-40LB cells were sorted and labeled with CellTracker Orange CMTMR fluorescent dye (Invitrogen, USA) and then separately injected intravenously to a mouse (Balb/c, 8–12 weeks old) together with 10 μg of AF488 anti-mouse MAdCAM-1 (Biolegend mAb MECA-367, USA) to identify high endothelial venules (HEVs). Under anesthesia, to stain the IgA+ cells for GC identification, 1 μg of AF647 anti-mouse IgA (Southern Biotech, USA) was directly injected to a PP of the same mouse transferred with iGB cells. The localization of transferred labeled iGB cells was observed under an LSM880 microscope (Carl Zeiss, Germany) and analyzed with ZEN2009 software (Carl Zeiss, Germany).
3
Cell Adhesion Assay for HUVEC-Monocyte Interaction
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Cell adhesion assay was performed according to the previously described procedure 56 . HUVECs were seeded on 0.1% gelatin-coated cover slides and were subjected to the indicated treatments. VCAM-1 neutralizing antibody (Abcam, #ab134047, 1:200) was added 30 min before FSH treatment. Human monocytic U937 cells (ATCC CRL-1593.2; Manassas, Virginia, USA) were incubated with CellTracker Orange CMTMR fluorescent dye (0.25 μL/mL; Molecular Probes; Life Technologies) in RPMI-1640 for 30 min, centrifuged and resuspended in RPMI-1640 containing 10% FBS culture medium. Subsequently, fluorescently-labelled U937 cells (1000 cells/mL) were seeded over HUVECs and allowed to adhere for 1 h. The non-adherent cells were washed away and the adherent fluorescent cells were counted under a fluorescence microscope and expressed relative to the total number of cells per field.
4
Monocyte Adhesion to VCAM-1 and Endothelial Cells
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Cell culture plates were coated overnight with recombinant mouse VCAM-1/Fc chimera (2 μg/mL; R&D Systems; MN, USA) at 4°C, rinsed with PBS and saturated with 2% BSA for 1 h. Human and murine primary circulating monocytes or U937 cells, were incubated with CellTracker orange CMTMR fluorescent dye (0.25 μl/ml; Molecular Probes; Life Technologies) in RPMI-1640 for 30 min, centrifuged and resuspended in RPMI-1640 + 10% FBS culture medium. Fluorescently-labelled cells (1000 cells/well) were incubated with the VCAM-1/Fc chimera-coated plates for 30 min with or without CD29 blocking antibody (0.5 μg; BD Pharmingen, clone 9EG7). Non-adherent cells were washed twice with PBS, and the adherent fluorescent cells were counted under a fluorescence microscope.
To assess monocyte adhesion to endothelial cells, HUVEC cells were placed on 0.1% gelatin-coated cover slides. At 80% confluence, cells were exposed to arsenic for 72 h. Arsenic-exposed, fluorescently-labelled U937 cells (1000 cells/ml) were seeded over HUVEC and allowed to adhere for 1 h. The non-adherent cells were washed away, and the co-culture was stained with DAPI 1:2 into mounting media (Vectashield h-2000, Vector Laboratories: Immu-Mount, Thermo Scientific, respectively) and the adherent fluorescent cells were counted under a fluorescence microscope and expressed relative to the total number of cells per field.
To assess monocyte adhesion to endothelial cells, HUVEC cells were placed on 0.1% gelatin-coated cover slides. At 80% confluence, cells were exposed to arsenic for 72 h. Arsenic-exposed, fluorescently-labelled U937 cells (1000 cells/ml) were seeded over HUVEC and allowed to adhere for 1 h. The non-adherent cells were washed away, and the co-culture was stained with DAPI 1:2 into mounting media (Vectashield h-2000, Vector Laboratories: Immu-Mount, Thermo Scientific, respectively) and the adherent fluorescent cells were counted under a fluorescence microscope and expressed relative to the total number of cells per field.
5
Epithelial Infection by Cell-Associated HIV
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