CAIA mice were intravenously injected with control IgG, unmodified α-TNF, or CBP–α-TNF at a dose of 200 μg per mouse. On day 5, hind paws were harvested and digested in Dulbecco’s modified eagle medium supplemented with 2% FBS, collagenase D (2 mg/ml) and deoxyribonuclease I (40 μg/ml; Roche) for 30 min at 37°C. Single-cell suspensions were obtained by gently disrupting through a 70-μm cell strainer. Antibodies against the following molecules were used: anti-mouse CD45 (30-F11, BD Biosciences), F4/80 (T45-2342, BD Biosciences), Ly6G (1A8, BioLegend), Ly6C (HK1.4, BioLegend), and CD11b (M1/70, BioLegend). Fixable live/dead cell discrimination was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer’s instructions. Following a washing step, cells were stained with specific antibodies for 20 min on ice. Flow cytometric analyses were done using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).
Fixable viability dye efluor 455
Manufactured by Thermo Fisher Scientific
Fixable Viability Dye eFluor 455 is a fluorescent dye used to identify dead cells in flow cytometry experiments. It binds to proteins in dead cells, allowing them to be distinguished from live cells during analysis.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase d, by Roche (2 mentions)
Cd45 30 f11, by BD (2 mentions)
Nk1.1 pk136, by BD (2 mentions)
Cd11b m1 70, by BioLegend (1 mentions)
Anti mouse cd45 30 f11, by BD (1 mentions)
Ly6g 1a8, by BioLegend (1 mentions)
Ly6c hk1, by BioLegend (1 mentions)
Fortessa, by BD (1 mentions)
Deoxyribonuclease 1, by Roche (1 mentions)
Cd8α 53 6.7, by BD (1 mentions)
Cd3 145 2c11, by BD (1 mentions)
Cd4 rm4 5, by BD (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Dnase 1, by Roche (1 mentions)
Ack lysing buffer, by Quality Biological (1 mentions)
Cd11b m1 70, by BD (1 mentions)
Cd103 m290, by BD (1 mentions)
Red blood cell lysis buffer, by BD (1 mentions)
Cd11c hl3, by BD (1 mentions)
Ly6g 1a8, by BD (1 mentions)
3 protocols using fixable viability dye efluor 455
1
Characterizing Immune Cells in Arthritic Mice
2
Tumor Immune Cell Analysis in MMTV-PyMT Mice
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The MMTV-PyMT model was prepared as described above. Mice were treated on day 7 with aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg). Mice were euthanized on day 14. Cell suspensions were obtained from each tumor as described previously (9 (link)). Tumors were harvested and digested in DMEM supplemented with 2% FBS, collagenase D (2 mg/ml), and DNase I (40 μg/ml) (Roche) for 30 min at 37°C. Single-cell suspensions were obtained by gently disrupting the organs through a 70-μm cell strainer. Red blood cells were lysed with ACK lysing buffer (Quality Biological). Fixable live/dead cell discrimination was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer’s instructions. Following a washing step, cells were stained with specific antibodies for 20 min on ice before fixation. The following antibodies were used to stain the cells: CD3 (145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), CD8α (53-6.7, BD Biosciences), CD45 (30-F11, BD Biosciences), and NK1.1 (PK136, BD Biosciences). All flow cytometric analyses were done using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
3
Comprehensive Murine Lung Cell Analysis
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Whole lungs were removed and chopped with razor blades, incubated with type IV collagenase (Worthington) at 37 °C for 40 min, then homogenized through a 70-µm cell strainer (Falcon). Remaining red blood cells were lysed using 1× red blood cell lysis buffer (BD Biosciences). Cells were stained with Fixable Viability Dye eFluor® 455 (eBioscience). Anti-mouse immunophenotyping antibodies were diluted in FACS buffer (3% FBS, 2 mM EDTA, 1× PBS) to 5 µg per mL along with Fc block (anti-mouse CD16/CD32; 5 µg per mL, BD), and cells were stained for 30 min on ice in three groups (AMφ: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), and Siglec-F (E50-2440; BD); monocyte and neutrophil: Ly-6C (AL-21; BD), Ly-6G (1A8; BD), and CD11b (M1/70; BD); dendritic cell: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), MHC II (M5/114.15.2; BD), and CD103 (M290; BD); NK cells: CD3 (17A2; BD), NK-1.1 (PK136; BD), and CD49b (DX5; BD)). After the staining, cells were washed twice with FACS buffer and then fixed in 2% para-formaldehyde in FACS buffer for 15 min. Cell numbers were counted using AccuCount Fluorescent Particles (Spherotech). All data were collected on an LSR II flow cytometer (BD) and analyzed using FlowJo software.
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