Naïve T cell homing was examined as reported previously70 (link). Briefly, naïve T cells were labelled using CellTracker CMTMR and CMFDA probe (Life Technologies) according to the manufacturer's protocol, mixed in a 1:1 ratio and a total of 1 × 107 cells was injected into the tail vein of a wild-type mouse. The animals were killed after 3 h and the competitive recruitment to peripheral (axillary and inguinal) lymph nodes and the spleen was analysed via flow cytometry. For half of the conducted experiments, celltracker colours were exchanged to exclude dye toxicity effects.
Celltracker cmtmr
Manufactured by Thermo Fisher Scientific
CellTracker CMTMR is a fluorescent dye used for long-term cell labeling. It is a cell-permeant dye that freely diffuses into cells and gets converted to a cell-impermeant form, allowing for long-term labeling and tracking of cells.
Automatically generated - may contain errors
Lab products found in correlation
Cmfda, by Thermo Fisher Scientific (2 mentions)
Mathematica, by Wolfram (1 mentions)
Donor horse serum, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Escherichia coli lps, by Merck Group (1 mentions)
Bx50wi fluorescence microscope, by Olympus (1 mentions)
6 protocols using celltracker cmtmr
1
Naïve T Cell Homing Assay
2
T Cell Transmigration Assay
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To evaluate T cell transmigration, T cells labelled with Celltracker CMTMR and CMFDA (Life Technologies) were perfused over confluent, TNFα-activated SEND-1 cells (provided by Dr M. Gimbrone, Brigham & Women's Hospital, Boston, Massachusetts) and given time to adhere. After 5 min, 2 dyne per cm2 shear was applied and lymphocyte movement was recorded over 10 min. Migration velocity was calculated as the displacement over time of migrating cells and transmigration frequency was calculated as the percentage of adherent cells that completely transmigrated through the endothelial cell layer at any point during the observation period. For every other experiment, cell dyes were exchanged to account for differences in dye toxicity.
3
Dynamic Interaction Analysis of Macrophages and Cancer Cells
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BMDMs and E0771-LG cells were labeled with CellTracker CMTMR and CMFDA (Invitrogen), respectively. To determine dynamic interaction between BMDMs and cancer cells, single cell suspension of each cells (104) were placed on Matrigel (5 mg/ml) and cultured for 1 h in αMEM supplemented with 10%FBS and 104U/ml CSF-1. Bright-field and fluorescent images (10 randomly selected fields in each well) were acquired every 10 min by fluorescence microscopy (Axiovert 200). Movies of BMDMs were tracked in Fiji ImageJ and tracks were analyzed in Mathematica (Wolfram) to determine the duration of interaction between BMDMs and cancer cells. To detect BMM binding to cancer cells, single cell suspension of each labeled cells (5 × 105) were mixed in v-bottom 96-well plates and cultured for 1 h in αMEM supplemented with 10%FBS and 104 U/ml CSF-1. The ratio of CMFTMR/CMFDA double-positive population was determined by flow cytometry. Based on a side-scattered light (SSC), we confirmed that >80% of double-positive fraction consists doublet of cells whereas <10% of single positive fraction does. To maintain binding activity of integrin, the cells were kept in Hepes buffer containing CaCl2 and MgCl2 during the analysis.
4
Quantifying HIV-1 Infection in Cocultured T Cells
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2 × 105 A3.01 T cells were inoculated with HIV-1NL4-3 (approximately 1.2 µg p24). Five days post-infection, the cells were stained with 3 µM CellTracker CMTMR (Invitrogen) for 15 min at 37 °C, washed with PBS, and further cultured for 1 day. The stained cells were washed with PBS and used as donor cells. 6 × 105 A3.01 T cells and 3 × 105 CMTMR-stained donor cells were either cocultured or cultured separately using transwell in the presence or absence of lnFRCs. After 2 days, T cells were fixed with 4% PFA/PBS, permeabilized with 0.1% Triton X-100, and immunostained with anti-HIV Gag-FITC (clone KC57; Beckman Coulter). The infected cells were analyzed by flow cytometry. T cells were distinguished from lnFRCs by forward and side scatter, as T cells were much smaller and less granular than lnFRCs.
5
Adhesion of C2C12 Myoblasts to Actuating Hydrogels
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C2C12 myoblasts were maintained in culture
at 37 °C with 5% CO2 concentration in growth media
(DMEM (Gibco) supplemented with 10% fetal bovine serum (Corning) and
1% penicillin–streptomycin (Corning)), and passaged no more
than 15 times before use in experiments. Glass slides with hydrogels
were fitted into metal imaging chambers and cells were plated at a
density of 60,000 cells/chamber in growth media and allowed to attach
overnight before experiments were conducted. To validate cell adhesion
to actuating hydrogels, gels were formed with and without laminin,
and adhered cells were visualized with 10 μM CellTracker CMTMR
(Invitrogen). The number of attached cells and cell spreading area
were quantified.
For experiments longer than 1 day, cell media
was changed daily, using growth media until cells reached ∼80%
confluency, at which point media was changed to DMEM supplemented
with 2% donor horse serum (Corning) and 1% penicillin–streptomycin.
at 37 °C with 5% CO2 concentration in growth media
(DMEM (Gibco) supplemented with 10% fetal bovine serum (Corning) and
1% penicillin–streptomycin (Corning)), and passaged no more
than 15 times before use in experiments. Glass slides with hydrogels
were fitted into metal imaging chambers and cells were plated at a
density of 60,000 cells/chamber in growth media and allowed to attach
overnight before experiments were conducted. To validate cell adhesion
to actuating hydrogels, gels were formed with and without laminin,
and adhered cells were visualized with 10 μM CellTracker CMTMR
(Invitrogen). The number of attached cells and cell spreading area
were quantified.
For experiments longer than 1 day, cell media
was changed daily, using growth media until cells reached ∼80%
confluency, at which point media was changed to DMEM supplemented
with 2% donor horse serum (Corning) and 1% penicillin–streptomycin.
6
Visualizing Treg-DC Interactions In Vivo
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BMDCs were prepared from bone marrow of C57BL/6 mice and transfected with either control or CCL22 siRNA. Subsequently, DCs were pulsed with 1 µg/ml OVA323–339 peptide and labeled for 20 min at 37°C with 10 mM 5- and 6-([(4-chloromethyl) benzoyl] amino) tetramethylrhodamine (CellTracker CMTMR; Invitrogen) or 7-amino-4-chloromethylcoumarin (Cell Tracker CMAC; Invitrogen). Control siRNA- and CCL22 siRNA-treated DCs (both 106) were coinjected in 20 µl IMDM (with 10% FCS) containing 10 ng Escherichia coli LPS (Sigma) into the right hind footpad of C57BL/6 OT-II-Foxp3-GFP mice. 18 h after injection of DCs, animals received 100 µg anti-CD62L antibody (Mel-14) to inhibit lymph node homing during imaging, and the right popliteal lymph node was analyzed for 1 h by two-photon intravital imaging with an Olympus BX50WI fluorescence microscope equipped with a 20-fold magnification, 0.95-NA objective (Olympus) as described (Mempel et al., 2004 (link)). Image data collection was repeated every 20 s for 1 h. The 4D image dataset was processed using Imaris software (Bitplane) to create sequential 2D maximum-intensity projections. The absolute number of T reg contacts per DC over the time period of 1 h was quantified by individual inspection of each time point. Cellular interactions that were <2 min or incompletely depicted spatially or temporally were excluded from the analysis.
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