Atplite one step luminescence assay kits

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.

4T1 cells were plated in 12-well plates at the density of 1 × 10⁵ cells/well. After one night, the medium was removed and replenished with new cell culture medium containing Lipo-SM/Chol, free CPT, Onivyde^® or Camptothesome at the concentration of 4 μM or 15 μM (CPT or irinotecan) for 20 h, respectively. Supernatants were collected to measure the released HMGB1 or ATP concentrations by using an HMGB1 ELISA kit (IBL International GmbH, #ABIN6574155) or ATPlite one-step luminescence assay kits (PerkinElmer, #6016736) following the manufacturers’ instructions, respectively. Cells were trypsinized and harvested, washed with 4 °C PBS twice, and 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. The cells were washed with PBS (4 °C) twice, resuspended in staining buffer (400 μL) and then analyzed by a BD FACSCanto^™ II flow cytometer (BD Bioscience). The data were presented as normalized calreticulin ratio versus the no treatment control group.

The divided tumors were homogenized by bead beating in PBS (1 mL PBS/100 mg tissue) at 4 °C. The supernatants were collected for ELISA tests after centrifuging at a speed of 15,000 rpm/min for 10 min. The measurement of IFN-γ and ATP were according to the manufacturer’s protocol of IFN-γ (LSBio, LS-F3414) and ATPlite one-step luminescence assay kits (PerkinElmer, #6016736), respectively.