Clear u bottom 96 well plates

Ricin binding to cell surfaces was performed as described [26] (link). In brief, THP-1 cells were collected by gentle, low speed centrifugation (5 min at 400×g). The resulting cell pellets were suspended to ∼5 ×10⁶ cells per mL and then seeded (200 µl/well) into clear U-bottom 96-well plates (BD Bioscience, San Jose CA). FITC-labeled ricin (3 µg/mL) was mixed with Abs or lactose (30 mg/mL) for 30 min on ice in the dark prior to being added to THP-1 cells. Cells were then washed twice with PHEM buffer to remove unbound toxin:Ab complexes and fixed with 4% paraformaldehyde (PFA) in PHEM for 15 min. Ricin binding to the surfaces of THP-1 cells was measured using FACS Calibur flow cytometer (BD Bioscience, San Jose CA). A minimum of 10,000 events was analyzed per sample.

THP-1 cells were collected by centrifugation (5 min at 400 x g) and the resulting cell pellets were suspended to 5x10⁶ cells per ml, as described [25 (link)]. The cells were seeded (200 μl/well) into clear U-bottom 96-well plates (BD Bioscience; San Jose, CA). Ricin-FITC was mixed with antibodies and incubated in the dark for 30 min on ice. Cells were washed with PHEM buffer [60 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9] to remove unbound ricin:antibody complexes, then incubated with 0.1 M lactose to remove surface bound toxin. The cells were re-suspended in sorting buffer [1x PBS, 25 mM HEPES, 1 mM EDTA] prior to analysis using a FACS Calibur flow cytometer (BD Bioscience). A minimum of 10,000 events was analyzed per sample.

THP-1 cell cytotoxicity assays were done as described [40] (link). Briefly, THP-1 cells were spun (5 min at 400×g) and adjusted to ∼5 ×10⁴ cells per mL and seeded (100 µl/well) into clear U-bottom 96-well plates (BD Bioscience, San Jose CA) and allowed to grow overnight. The next day, THP-1 cells were spun to remove medium and were then treated with ricin (0.01 µg/mL; 154 pM), ricin:Ab mixtures, or medium alone for 2 h at 37°C. Cells were then subjected to centrifugation and washed to remove non-internalized toxin or ricin:Ab mixtures. Fresh medium was added to the wells and allowed to incubate for 48 h. Cell viability was determined using CellTiter-Glo after the content of each plate was transferred into white bottom 96-well plates. In order to address post-attachment experiments, THP-1 cells were kept on ice at 4°C, to allow for ricin attachment but prevented internalization of the toxin prior to antibody treatment. The cytotoxicity assay was performed as described above but cells were kept on ice until they were transferred to 37°C for the 48 h incubation period. All samples were performed in quadruplicate and 100% viability was defined as the average value obtained from wells in which cells were treated with medium only.