A total of 1 × 105 CT26 or MC38 cells were plated in 12-well plates. After 12 h, the cell culture medium was removed and replenished with free CPT, Onivyde or Camptothesome containing media at the 1 μM or 5 μM (CPT or Onivyde) for 20 h. Supernatants were collected to measure the released HMGB-1 or ATP concentrations by using an HMGB-1 ELISA kit (IBL International GmbH, #ABIN6574155) or ATPlite one-step luminescence assay kits (PerkinElmer, #6016736) following the manufacturers’ instructions, respectively. To determine the cellular surface calreticulin expression by flow cytometry, cells were trypsinized and washed with cold PBS first, then stained with an Alexa Fluor® 647-conjugated anti-calreticulin antibody (Abcam, ab196159, 1/500) in staining buffer (eBioscience, #00-4222-26) at 4 °C for 30 min. After being washed with PBS (4 °C) twice, the cells were suspended in 400 μL of staining buffer and analyzed by a BD FACSCanto™ II flow cytometer (BD Bioscience). The data were presented as fold-increase in mean positive percentage ratio compared to the no treatment control group.
Alexa fluor 647 conjugated anti calreticulin antibody
Manufactured by Abcam
Alexa Fluor® 647-conjugated anti-calreticulin antibody is a laboratory reagent used for the detection and quantification of calreticulin protein in biological samples. The antibody is conjugated with the Alexa Fluor® 647 fluorescent dye, which enables visualization and analysis of the target protein using techniques such as flow cytometry, immunofluorescence, and Western blotting.
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2 protocols using alexa fluor 647 conjugated anti calreticulin antibody
1
Quantification of Immunogenic Cell Death
2
Calreticulin Expression in Tumor Cells
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Cell membrane expression of calreticulin by CT26 and MCA205 cells after incubation with AgNP for 48 h was evaluated using flow cytometry. This timepoint was selected since the maximum effect in the anti-proliferative activity was noted at 48 h. For this assay, both Alexa Fluor ® -647-conjugated anti-calreticulin antibody (Abcam, Cambridge, UK) and APC/Cy7-conjugated anti-Zombie NIR cell death marker antibody (Biolegend, San Diego, CA) were employed. Here, cells were cultured in 12-well plates (at density of 5 × 10 5 /well). After 48 h of treatment with the test agent (or controls), the cells were removed, placed on ice, and then stained under dark conditions (following manufacturer protocols). After staining and repeated washings with flow cytometry staining buffer (containing 2% fetal bovine serum and 1% penicillin/streptomycin in phosphate-buffered saline [PBS, pH 7.4]), the cells were acquired in a Cytoflex S cytometer (Beckman Coulter, Santa Clara, CA) and analyzed using Flow Jo software (v.10.8, Ashland, OR) . For all samples, a total of 50 000 events was acquired. The assay was repeated twice in independent experiments. Samples were evaluated each time in triplicate.
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