MB49-EpCAM-OVA cells were labeled with the fluorescent dye PKH67 (Sigma-Aldrich), heat-shocked at 45°C for 10 min to induce necrosis, followed by incubation at 37°C overnight. The heat-shocked MB49-EpCAM-OVA cells were then co-cultured with hCD40tg DCs labeled with CellTracker Deep Red (Invitrogen) and CellTrace Violet-labeled OT-1 T cells. Images were captured using a Cytation 5 live cell imager.
Celltracker deep red
Manufactured by Thermo Fisher Scientific
Sourced in United States
CellTracker Deep Red is a fluorescent dye used to label living cells. It passively diffuses into cells and becomes trapped in the intracellular environment, allowing for long-term cell tracking and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Celltracker green cmfda, by Thermo Fisher Scientific (10 mentions)
Celltracker green, by Thermo Fisher Scientific (5 mentions)
Celltrace violet, by Thermo Fisher Scientific (4 mentions)
Celltracker blue, by Thermo Fisher Scientific (3 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
Celltracker red cmtpx, by Thermo Fisher Scientific (3 mentions)
Pkh67, by Merck Group (2 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
96 well ultra low attachment plate, by Fujifilm (2 mentions)
Celltracker red, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (2 mentions)
Cytochalasin d, by Cayman Chemical (2 mentions)
Matrigel, by Corning (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Annexin 5 bv421, by BD (2 mentions)
Celltracker blue cmac, by Thermo Fisher Scientific (2 mentions)
Celltracker orange cmtmr, by Thermo Fisher Scientific (2 mentions)
Fluoview fv1000, by Olympus (2 mentions)
95 protocols using celltracker deep red
1
Necrotic Tumor Antigen Presentation
2
Reticulocyte-Normocyte Co-culture Assay
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Enriched reticulocytes and normocytes were resuspended to 1% hematocrit with PBS and stained with CellTracker Deep Red (at 2 µM; Invitrogen #C34565) and CellTrace Oregon Green (at 15 µM; Invitrogen #C34555), respectively. After 30 mins of staining at 37°C, warm FBS was added to stop the staining reaction. Cells were then washed twice with media, with another 10 mins incubation during the second wash. Stained reticulocytes and normocytes were mixed to the preferred ratios (10:90, 30:70, 50:50, 70:30, or 90:10) and resuspended in parasite media to 2% hematocrit. Enriched late-stage parasites were then added at around 5-10% final parasitemia and the cells were incubated at 37°C for 12 hours. At Time = 0 hr (i.e. after the addition of parasites), aliquots of samples were mixed with PBS and stained with 8 µM Hoechst for 15-20 mins. 300 µL PBS was then added to quench the reaction and the samples were immediately acquired. The staining and flow cytometry acquisition was repeated at Time = 12 hr. This 12-hour incubation allows sufficient time for the trophozoites and schizonts to develop and release merozoites, and yet not too long until a second round of invasion occurs [e.g. the asexual cycle of different P. yoelii strains vary between 18-24 hours (Killick-Kendrick and Warben, 1968 (link); Gautret et al., 1994 (link))] or for the target reticulocytes to mature into normocytes.
3
Isolation and Expansion of Myeloid-Derived Suppressor Cells and Macrophages
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Primary bone marrow-derived MDSCs and MΦs were prepared from C57BL/6, MyD88−/−, or TLR2−/− mice as previously described (19 (link), 22 (link), 56 (link)). MDSCs were expanded for 4 days in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1% l- glutamine, 1% HEPES, 1% antibiotic-antimitotic, 50 μM beta-mercaptoethanol, 40 ng/ml GM-CSF, and 40 ng/ml G-CSF with 40 ng/ml IL-6 added at day 3 of culture. Following expansion, MDSCs were purified using an anti-Ly6G microbead kit (Miltenyi Biotec). MΦs were propagated for 7 days in RPMI-1640 supplemented with 10% FBS, 1% l -glutamine, 1% HEPES, 1% antibiotic-antimitotic, 50 μM beta-mercaptoethanol, and 10% conditioned medium from L929 fibroblasts as a source of macrophage colony-stimulating factor (M-CSF) (28 (link), 57 (link)). For visualizing MΦ invasion into biofilm by confocal microscopy, MΦs were stained with CellTracker deep red (1 μM; Invitrogen) according to the manufacturer’s instructions.
Human monocytes were obtained from healthy human donors by the UNMC Elutriation Core Facility by countercurrent centrifugal elutriation, in full compliance and with approval of the Institutional Review Board (IRB). Cells were cultured at 1 × 106 cells/ml in RPMI-1640 supplemented with recombinant human M-CSF, 10% human serum, and 1% antibiotic-antimitotic for 7 days until harvest for experiments.
Human monocytes were obtained from healthy human donors by the UNMC Elutriation Core Facility by countercurrent centrifugal elutriation, in full compliance and with approval of the Institutional Review Board (IRB). Cells were cultured at 1 × 106 cells/ml in RPMI-1640 supplemented with recombinant human M-CSF, 10% human serum, and 1% antibiotic-antimitotic for 7 days until harvest for experiments.
4
Immunostaining of NLV-specific CD8+ T cells
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PBMC were stimulated with 1 μg/ml of the HLA-A2-restricted immunodominant peptide CMVpp65495-503 (NLV) and expanded in a short-term culture for 8d as described above. On d8 CD8+ T cells were isolated using a negative CD8+ T cell isolation kit (Miltenyi Biotech) according to the manufacturer’s guidelines. T2 cells were pulsed with 1 μg/ml NLV peptide to function as APC.
APC were labelled with Cell Tracker Deep Red (Invitrogen) according to manufacturer’s instructions. CD8+ T cells and APC were resuspended in cRPMI with 1% FBS in a 1:1 ratio and incubated on poly-L-lysine coated coverslips for 30 min at 37°C. Cells were then fixed at 4°C in PBS with 4% formaldehyde and 1% bovine serum albumin (BSA) followed by a staining with 10 μg/ml CTB-FITC for 30 min at 4°C in cold PBS with 1% BSA and 0.01% NaN3. The cells were then permeabilised with 0.1% Triton in PBS with 1% BSA and stained with 10 μg/ml anti-CD3 epsilon mAb (UCHT1, Biolegend) followed by anti-mouse IgG1-AF546 and coverslips mounted with Prolong Gold anti-fade (ThermoFisher). Images were acquired using the DeltaVision ELITE Image Restoration Microscope (Applied Precision) coupled to an inverted Olympus IX71 microscope and a CoolSNAP HQ2 camera, deconvolved with softWoRx 5.0 and processed using Huygens Professional v4.0 and Adobe Photoshop CC 2018.
APC were labelled with Cell Tracker Deep Red (Invitrogen) according to manufacturer’s instructions. CD8+ T cells and APC were resuspended in cRPMI with 1% FBS in a 1:1 ratio and incubated on poly-L-lysine coated coverslips for 30 min at 37°C. Cells were then fixed at 4°C in PBS with 4% formaldehyde and 1% bovine serum albumin (BSA) followed by a staining with 10 μg/ml CTB-FITC for 30 min at 4°C in cold PBS with 1% BSA and 0.01% NaN3. The cells were then permeabilised with 0.1% Triton in PBS with 1% BSA and stained with 10 μg/ml anti-CD3 epsilon mAb (UCHT1, Biolegend) followed by anti-mouse IgG1-AF546 and coverslips mounted with Prolong Gold anti-fade (ThermoFisher). Images were acquired using the DeltaVision ELITE Image Restoration Microscope (Applied Precision) coupled to an inverted Olympus IX71 microscope and a CoolSNAP HQ2 camera, deconvolved with softWoRx 5.0 and processed using Huygens Professional v4.0 and Adobe Photoshop CC 2018.
5
Tumor Cell and Monocyte Perfusion Assay
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Tumor cells were resuspended at a concentration of 1 × 106 mL−1 in culture medium. Similar to dextran perfusion as described above, a 20 μL tumor cell suspension was perfused into the MVNs and incubated for 15 min. Tumor cell medium was aspirated from the media channels to remove the unattached cells, and Vasculife was replenished.The devices were then ready for imaging. To form tumor clusters to mimic circulating tumor clusters, tumor cell suspension was seeded in a 96-well ultra-low attachment plate (Wako Chemicals USA) at 2–4 cells per well. 24 h later, the tumor clusters were perfused. Monocytes were isolated from healthy donor’s blood by the monocyte core at MIT, and followed by CellTracker Deep Red (Invitrogen) staining. The experiments with primary blood cells were approved by the Committee on the Use of Humans as Experimental Subjects. After washing, monocytes were suspended at a concentration of 1 × 106 mL−1 in Vasculife and perfused.
6
High-Content Imaging of Apoptosis
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Assays were performed as for IncuCyte assays, with the exception that Hoechst stain (ThermoFisherScientific) was added for nuclear discrimination and apoptosis was imaged using the Opera Phenix High Content Screening System and analyzed with the Harmony High‐Content Imaging and Analysis software (PerkinElmer, Waltham, MA). Percent cytolysis was calculated from three‐color images normalized to the total number of cells in each well at each time point.
For assays with mixed target‐cell populations, PLC/PRF/5‐A2B2M were stained with CellTracker Deep Red, HepG2‐A2B2M with CellTracker Red, and pan T cells with CellTrace Violet (Invitrogen).
For assays with mixed target‐cell populations, PLC/PRF/5‐A2B2M were stained with CellTracker Deep Red, HepG2‐A2B2M with CellTracker Red, and pan T cells with CellTrace Violet (Invitrogen).
7
Treg-Mediated Tumor Modulation
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Spleens, tumors and tumor DLNs were harvested from MOC2 tumor-bearing DEREG mice 8 days post-treatment with 10 Gy RT. Samples were processed to single cell suspension in same method as above flow cytometry samples. Total T cells and Tregs were first isolated with an EasySep Mouse T Cell Isolation Kit (Stemcell) and then sorted using GFP signal encoded on the FOXP3 promoter on a Beckman Coulter MoFlo XDP70 cell sorter through The University of Colorado Cancer Center Flow Cytometry Shared Resource. MOC2 cells were cultured and irradiated with 10 Gy and 72 hours later were stained with Cell Tracker Deep Red (Invitrogen) before being combined with sorted Tregs with a 2:1 ratio of Tregs to MOC2 cells with the irradiated conditioned media. Tregs and MOC2 cells were incubated at 37°C for 4 hours in the presence of mouse IL-2 recombinant protein (Invitrogen) at 2 ug/mL and anti-CD137 (4-1BB, Bio X Cell) (Clone:3H3, Bio X Cell) or isotype equivalent control (rat IgG2a isotype control, antitrinitrophenol)(Clone:2A3, Bio X Cell) at 10 ug/mL. Samples were stained with a live/dead aqua viability stain kit (Invitrogen) for 30 min at 4°C and then analyzed on an Aurora spectral cytometer (Cytek Biosciences, Fremont, California, USA). Representative gating is shown in online supplemental figure 9 .
8
Mitochondrial Dynamics in Macrophage Activation
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hMDMs were differentiated at 30,000 cells per well in glass-bottom 96-well plates (Cellvis, P96-1.5H-N). Parallel plates were prepared for live-cell imaging and LDH + CellTiter Glo analysis. hMDMs were either left untreated or pre-treated with glycine (50 mM) and primed with 1 μg/mL LPS (E. coli serotype 0111:B4) in RPMI complemented with 1% fetal calf serum for 3 hr before stimulation with 20.7 μM of nigericin (Invivogen). Tetramethylrhodamine ethyl ester perchlorate (TMRE, 200 or 10 nM, Sigma-Aldrich) was added to cells before the plates were placed in a heated incubator (37°C, 5% CO2), and images were taken every 30 min over 18 or 22 hr (two independent experiments). In the second experiment, at the end of LPS priming, cells were labeled with CellTracker Deep Red (0.5 μM Invitrogen) for 15 min and washed before addition of fresh medium with TMRE (10 nM) with or without LPS, nigericin, and glycine. In parallel plates treated identically, supernatants were harvested and assayed for LDH, and cellular ATP was measured using CellTiterGlo (Promega) at 0, 2, 6, and 18/22 hr.
9
Tumor Cell and Monocyte Perfusion Assay
10
Infection of THP-1 Cells with Tachyzoites
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Infected HFFs were lysed and filtered through a 3-μm pore-sized membrane to remove host cell debris. Prepared tachyzoites were added to THP-1 cells at a density of 106 cells/mL (parasite/cell ratio 2). After incubation for 24 h at 37°C in an atmosphere containing 5% CO2, infection was confirmed under the fluorescence microscope. Then, supernatants were separated by centrifugation at 1000g for 10 min and filtration through a 0.22-μm pore-sized filter. The collected supernatants were stored at -20°C. Heat-inactivated tachyzoites were prepared after incubation at 56°C for 30 min as previously described [14 (link), 15 (link)]. For immunocytochemistry, THP-1 cells were labeled with CellTracker™ Deep Red (Invitrogen) prior to the addition of the tachyzoites.
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