For DDR1 protein analysis, cells were washed in phosphate-buffered saline (PBS) and resuspended in 200 μL of PBS. Cells were then incubated with 5 μL of anti-DDR1 antibody conjugated with paraffin embedded (PE, PE anti-human CD167a, Biolegend, San Diego, CA; cat #334005) and 1.5 μL of ChromPure Rat IgG Whole Molecule (Jackson ImmunoResearch #012-000-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h shaking at 4°C. Following incubation, cells were washed and resuspended in 500 μL of PBS and analyzed on flow using FACScalibur (BD Biosciences, San Jose, CA).
Chrompure rat igg whole molecule
Manufactured by Jackson ImmunoResearch
ChromPure Rat IgG Whole Molecule is a laboratory reagent used for research purposes. It is purified rat immunoglobulin G (IgG) that can be used in various immunological applications.
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Lab products found in correlation
Facscalibur, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Anti cd16 cd32 2.4g2, by BioXCell (1 mentions)
Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mhc 2 m5 114.15.2, by BD (1 mentions)
2 protocols using chrompure rat igg whole molecule
1
DDR1 Protein Expression Analysis
2
Multicolor Flow Cytometry of Immune Cells
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Dead cells were stained using LIVE/DEAD® fixable dead cell stain kit (Invitrogen) as described by the manufacturer. Fluorochrome-coupled antibodies recognizing mouse CD3 (clone 145-2C11), CD4 (RM4-5), CD11b (M1/70), CD11c (N418), CD19 (MB19-1), CD25 (PC61.5), CD44 (IM7), CD49b (DX5), CD62L (MEL-14), CD86 (GL1), CD90.2 (53-2.1), iNOS (C-11), and MHCII (M5/114.15.2) were purchased from either BD Biosciences, BioLegend, eBioscience or Santa Cruz Biotechnology. Specific antibody staining was performed at 4 °C in the dark for 15 min (surface staining) or 30 min (intracellular staining using Foxp3/Transcription Factor Staining Buffer Set from eBioscience). Spleens were stained in a total volume of 100 μl, and lung samples in 200 μl. To reduce unspecific antibody binding, surface and intracellular staining were performed in the presence of anti-CD16/CD32 (2.4G2, BioXCell) antibodies and ChromPure rat IgG whole molecule (Jackson ImmunoResearch), respectively, and using predetermined optimal antibody dilutions. Carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) staining (10 μM) was performed at 2 × 107 cells/ml of PBS for 2 min and 45 s at room temperature. Reaction was stopped by adding a tenfold volume of complete RPMI 1640 medium (Invitrogen) containing 10% fetal calf serum (cRPMI) followed by two washing steps.
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