Mouse hind limbs were collected and bisected by cutting through the knee joint. Muscle and connective tissues of both the femur and tibia were removed by scraping the diaphysis of the bone. After cleaning, bones were rinsed in ice-cold 70% ethanol for 10-15 min and washed in cold PBS for 5 min for three times. The femur and tibia were cut just at the end of the marrow cavity and the marrow was sluiced out by RPMI 1640 (Invitrogen) containing 10% FBS and 2 mM EDTA followed by filtering through 100 μm filter. Cells were centrifuged at 2 000× g for 10 min and red blood cells were lysed with RBC lysis buffer (Roche). Washed and collected cells from previous step were purified by 40%/80% percoll, and stained sequentially with Violet Dye and antibody cocktail for flow cytometry analysis. The antibody cocktail for different lineage markers included CD3e-PECy7 (eBioscience, 25-0031), CD19-PECy7 (eBioscience, 25-0193), CD11b-PECy7 (eBioscience, 25-0112), Ly6G-PECy7 (eBioscience, 25-5931), Ter119-PECy7 (eBioscience, 25-5921). The cells were stained spontaneously with an antibody against Kit/CD117-APC (eBioscience, 17-1171, 1:200).
Cd3e pe cy7
Manufactured by Thermo Fisher Scientific
The CD3e PE-Cy7 is a fluorescently labeled antibody used for the detection and analysis of CD3e-positive cells. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b pe cy7, by Thermo Fisher Scientific (4 mentions)
Cd19 pecy7, by Thermo Fisher Scientific (2 mentions)
Ter119 pecy7, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Rbc lysis buffer, by Roche (1 mentions)
Ly6g pecy7, by Thermo Fisher Scientific (1 mentions)
Cd44 apc, by Thermo Fisher Scientific (1 mentions)
Cd8a pe, by Thermo Fisher Scientific (1 mentions)
Macsquant flow cytometer, by Miltenyi Biotec (1 mentions)
Macsquantify software, by Miltenyi Biotec (1 mentions)
Cd62l fitc, by Thermo Fisher Scientific (1 mentions)
Wheatley dounce homogenizer, by Thermo Fisher Scientific (1 mentions)
40 μm nylon mesh filter, by Thermo Fisher Scientific (1 mentions)
Fc block cd16 32, by Thermo Fisher Scientific (1 mentions)
Cd4 apc cy7, by Thermo Fisher Scientific (1 mentions)
Cd45 eflour 450, by Thermo Fisher Scientific (1 mentions)
F4 80 apc, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd16 cd32 fc block, by Thermo Fisher Scientific (1 mentions)
Cd45 apc cy7, by BioLegend (1 mentions)
Cd4 pe, by BioLegend (1 mentions)
5 protocols using cd3e pe cy7
1
Isolation and Purification of Bone Marrow Cells
2
Flow Cytometry Analysis of T Cell Subsets
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Following the removal of the olfactory bulb at the indicated time points, tissue was placed in a 2 ml Wheatley Dounce Homogenizer (Fisher Scientific) with 2 ml DMEM media supplemented with high glucose, L-glutamine, and pyruvate (Life Technologies) and 10% FBS. Single cell suspensions were created and processed as previously described [21 (link)]. Briefly, 1/10 the sample homogenate was filtered using a 40-μm nylon mesh filter (Fisher), was pre-incubated with 0.8 μg Fc block (CD16/32) (eBioscience) and then was immunolabeled in 1% FBS/BSA. Total T cells were stained for CD45 eFlour 450 (clone 30-F11), CD3e FITC (clone 145-2C11), CD8a PE (clone 53-6.7), and CD4 APC (clone GK1.5) (all eBioscience). Effector (T-EM) and central memory (T-CM) cells were identified by CD45 eFlour 450, CD3e PE-Cy7, CD4 APC-Cy7, CD8a PE, CD44 APC, and CD62L FITC all from eBioscience as described [21 (link)]. HSV-1-specific T cells were identified by CD3 eFluor 450, CD8a FITC, and either gB-PE, ICP8-A488, or RRI-A488 tetramers (provided by the NIH tetramer facility) as previously described [21 (link)]. All samples were analyzed with the MacsQuant flow cytometer and MacsQuantify software (Miltenyi Biotec).
3
Multicolor Flow Cytometry Analysis
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Blood, bone marrow, BAL, and lung cells were counted using an automated cell counter (Nexcelcom Bioscience, Lawrence, MA). Cells were incubated with Fc block (anti-mouse CD16/CD32; eBioscience) in 100 μl of FACS buffer (PBS, 0.5 % EDTA) followed by the antibody panels below. Labeled cells were analyzed using BD FACSCanto RUO (BD Biosciences, San Jose, CA) and the FlowJo data analysis software (Ashland, OR). Experiments represent an average of n = 5/group with up to 3 replicates. Antibodies used included: anti-mouse CD62L-APC, ICAM1-FITC, Ly6G-FITC, Ly6G-PerCP/Cy5.5, LFA-1-PE, CD45-APC/Cy7, CD4-PE, CD8-PB, CXCR2-PerCP/Cy5.5, Gr1-PB, Ly6C-PB (BioLegend, San Diego, CA), CD11C-APC, CD11b-PECy7, CD3e PE/Cy7, CD3e-PerCP/Cy5.5, CD71-PE, F4/80-APC (eBioscience).
4
Cardiomyocyte Viability and Characterization
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Dissociated cardiomyocytes were first stained with the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Life Technology, L34955) according to the manufacturer's instruction. 500 μl isolation buffer (2 mM EDTA and 0.5% BSA in PBS) was added to wash the cells, followed by centrifugation for 3 min at 20× g. These cardiomyocytes were subsequently stained with antibody mixture (Lin) containing CD3e PE-Cy7 (eBioscience, 25-0031), CD19 PE-Cy7 (eBioscience, 25-0193), CD11b PE-Cy7 (eBioscience, 25-0112), CD11c PE-Cy7 (eBioscience, 25-0114), Ly-6G (Gr-1) PE-Cy7 (eBioscience, 25-5931), Ter-119 PE-Cy7 (eBioscience, 25-5921) at a dilution of 1:200 for 30 min at 4 °C. Finally, the cells were re-suspended in 500 μl isolation buffer. The stained cells were analyzed using a FACS Aria II Flow Cytometer (BD Bioscience), and the raw data processed by Flowjo (TreeStar) according to protocols described previously13 (link).
5
Multiparametric flow cytometry analysis
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After red blood cell lysis, stainings were performed in the presence of 10 μl/mL of FcR blocking reagent (Miltenyi) in PBS with 2% FBS. For flow cytometry analysis, acquisition and data analysis were conducted on a LSR Fortessa cytometer (BD Biosciences) and FlowJo v10.7 software. For Next Generation Sequencing, cells were sorted using Influx Cell Sorter (BD Biosciences) and BD FACSTM v1.2.0.142 software (BD Biosciences). We used the following antibodies from BD Biosciences or eBiosciences: CD11b-PE-Cy7, CD19-PE-Texas Red, CD25-APC, CD253-APC, CD274-PE, CD3e-PE-Cy7, CD4-efluor 450, CD45-BV711, CD8b-FITC, F4/80-Alexa-eFluor 647, FoxP3-PE, Granzyme B-PE, Ly6G-FITC, MHC Class II-APC efluor 780, NK1.1-FITC, PD1-APC. Live and dead cells were distinguished using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life technologies). The gating strategy is shown in Fig. S1 .
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