mFX and/or radiation-induced tumor cell death was assessed using Annexin V-7AAD apoptosis kit (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. As previously described (25 (link)), cell surface calreticulin or ERp57 was detected by staining of anti-calreticulin antibody (1:1000, ab4109, Abcam) or anti-ERp57 antibody (1:1000, ab10287, Abcam) for 30 min at 4 °C in the dark. Cells were washed with PBS containing 5% FBS, followed by AF488-labeled 2nd antibody for 30 min at 4 °C in the dark. Cells were washed again with PBS containing 5% FBS, and analyzed by flow cytometry. Cell culture supernatant was assayed for extracellular HMGB1 by ELISA (Fisher Scientific) according to the manufacturer’s instructions.
Ab4109
Manufactured by Abcam
Ab4109 is a primary antibody that targets the ABC transporter family. It is designed for use in various laboratory applications, including Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
2 protocols using ab4109
1
Quantifying Tumor Cell Death and Immunogenic Markers
2
Immunohistochemical Analysis of Tumor Markers
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The cleaved caspase-3, HMGB1, or calreticulin-positive cells were determined by immunohistochemical staining as described previously.21 (link) Briefly, the frozen sections of tumor tissue from KCKO-Luc or KP2.1-Luc bearing mice were stained with primary antibodies, including cleaved caspase-3 (#9664S, Cell Signaling Technology), anti-HMGB1 antibody (ab18256, Abcam), or anti-calreticulin antibody (ab4109, Abcam), followed by horseradish peroxidase-labeled secondary antibody staining. 3,3′-Diaminobenzidine (DAB) was applied as substrate and hematoxylin as counterstaining. Positive cells were enumerated using a computerized Olympus DP80 imaging system. For cleaved caspase-3 or HMGB1 staining, 10 randomly selected fields (×400, magnification) of each tumor tissue section were enumerated, and the means were reported. The calreticulin expression in tumor tissue was examined and scored by a licensed pathologist (blinded): 0, no staining; 1, low intensity of staining, or <25% of tumor cells were positive; 2, medium level of intensity of staining, or >25% and<50% of tumor cells are positive; 3, the high-level intensity of staining, or >50% and<75% of tumor cells are positive; or 4, the maximum high-level intensity of staining, or >75% of tumor cells are positive.
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