Lsrii 4 laser

CD11c⁺ BMDCs were plated in a 24-well plate at a density of 2 × 10⁶ cells/ml and incubated with 2 μg/ml scFvMTBHsp70 (105 kDa), 1.3 μg/ml MTBHsp70 (70 kDa), 1 μg/ml LPS equivalent to 10³ EU/ml endotoxin (InvivoGen, San Diego, CA), or 0.1 ng/ml (0.1 EU/ml) LPS equivalent to endotoxin found in 2 μg/ml of proteins (since LPS level is less than 50 EU per mg of protein) for 24 h at 37°C in humidified atmosphere with 5% CO₂. Cells were then placed on ice, collected by vigorous pipetting, washed and stained with the following fluorophore-conjugated antibodies: anti-CD11c and anti-CD40 (eBioscience), anti-CD80 (BD Horizon), anti-CD86 and anti-MHC class II (I-A^q) (BD Pharmingen). Afterwards, the cells were analyzed on an LSRII 4 laser (BD Biosciences).

Single cell suspensions were prepared from spleens. Cells were plated in round-bottomed 96-well plates, pulsed with a validated CD8⁺ T-cell Her2/neu peptide (PDSLRDLSVF, 1 μg/ml; EZBiolab)
[25 (link),43 (link)], an in-house designed H2^d-restricted MSLN Ld1 peptide (IPLSYLCDF, 1 μg/ml; EZBiolab) that did not induce ovarian cancer specific T-cell response in H-2^q FVB mice, or medium alone for 72 hours when Golgi Plug (BD Bioscience) was added for the last 5 hours as previously described
[44 (link)], and then stained with fluorophore-conjugated anti-CD3, anti-CD4, anti-CD8, anti-IFNγ (BD Pharmingen), and anti-Granzyme B (eBioscience) antibodies. Cells were then analyzed on a LSRII 4 laser (BD Biosciences).

BR5FVB1 cells were harvested and treated with mitomycin C, and plated in a 96-well round-bottomed plate with 20 μg/ml scFvMTBHsp70 or 13 μg/ml MTBHsp70. After pre-incubation at 4°C for 1 h, CD11c⁺ BMDCs (ratio of tumor cells: DCs = 3: 1) were added to the wells and the plate was incubated at 37°C for 24 h. For generation of BR5FVB1 cell-primed T cells, we inoculated FVB/NJ mice by i.p. injection with 10⁷mitomycin C-treated BR5FVB1 cells and sacrificed the mice 60 days after the immunization according to the approved animal protocol. Splenocytes were then harvested, and T cells were isolated using the Pan T-Cell Isolation Kit II (MiltenyiBiotec). BR5FVB1 cell-primed T cells were then added to the wells at a DC/T-cell ratio of 1:20. After a 24-hour co-culture of BR5FVB1 cell-pulsed DCs with BR5FVB1 cell-primed T cells, the cells were harvested, washed and resuspended in PBS with 5% FBS, stained for CD3, CD4, CD8 and IFNγ, and analyzed on a LSRII 4 laser (BD Biosciences).