Isotype matched igg antibodies

iRTN-1c MCA205 cells were treated (or not) by 1 µM MTX, 150 µM CDDP, 0.3 µM TET, or CDDP+TET, for 24 h, prior to cell harvest and subsequent wash with ice cold PBS. After 30 min incubation with an anti-CRT antibody (Abcam) in cold blocking buffer (5% FBS, v/v in PBS) on ice, cells were further washed and incubated with AlexaFluor^®488-conjugated secondary antibody (Life Technologies) in blocking buffer (for 30 min). Cells were finally washed and maintained in cold PBS with 1 μg/mL PI and samples were analyzed by means of a FACS Calibur cytofluorometer (BD Biosciences). Isotype-matched IgG antibodies (Cell Signaling Technology) were used a negative staining control, and the analysis was performed exclusively on non-permeabilized (PI^-) cells. Data were statistically evaluated by means of the Cell Quest Software package (BD Biosciences).

Cells were harvested and washed with ice-cold PBS, then incubated with a CRT-specific antibody (Abcam) diluted in cold blocking buffer (5% FBS, v/v in PBS) for 30 min on ice, washed, and incubated with an AlexaFluor^® 488-conjugated antibody (Life Technologies Inc.) in blocking buffer (for 30 min). Thereafter, cells were washed, stained with 1 μg/mL PI in cold PBS for 5 min, and analyzed by means of a FACSCalibur cytofluorometer (BD Biosciences). Isotype-matched IgG antibodies (Cell Signaling Technology) were used as negative staining control. First line statistical analyses were performed by using the CellQuest™ software (BD Biosciences), upon gating on PI^- events characterized by normal forward and side scatter (living cells).