Cd45 ko

The flow-cytometry-based strategy used in this study involved 10 characteristic antibodies, including 8 against cell-membrane proteins (CD138-APC, CD38-APC750, CD19-ECD, CD45-KO, CD56-PC7, CD27-PB, CD81-APC700, and CD117-PC5; from Beckman Coulter, USA), and 2 against cytoplasmic proteins (Kappa-FITC and Lambda-PE; from Dako, Denmark), which have previously been described to be able to differentiate between abnormal and normal plasma cells via flow cytometry [20 (link)–22 (link)]. Sample preparation and detection were performed as previously described [17 ]. Briefly, 200 μL–5 mL BM aspirates [(2–20) × 10⁶ cells] were stained with the monoclonal antibodies for 30 min at 20–30 °C after lysing red-blood cells by using ammonium chloride and then washed with phosphate-buffered saline. For intracellular light-chain evaluation, cells were stained with anti-kappa and anti-lambda antibodies after adding membrane breakers, followed by washing and incubation. Flow-cytometry events were acquired and analyzed using a Navios Flow cytometer (Beckman Coulter, USA). One million nucleated cells were obtained each time, and if ≥ 20 abnormal plasma cells were detected, the result was considered positive.

ZIKV NS1 monoclonal antibodies were isolated from the memory B cell culture method in vitro [15 (link)]. Briefly thawed PBMCs derived from the two ZIKV-infected individuals (on day188 in patient1 and day66 in patient 2) were stained with a panel of fluorescence-labeled Abs (IgD-FITC, CD19-ECD, CD27-PC7, CD38-APCA750, IgM-PB, and CD45-KO Beckman Coulter). Memory B cells gated as IgD IgM - CD27 + CD38 low were sorted into 96-well culture plates (25 to 50 cells/well) and cultured with supplements as described previously [15 (link)]. After 10 days, the culture supernatants were screened for the presence of ZIKV NS1 binding mAbs using capture ELISA described in next paragraph. VH and VL sequences were obtained from positive B-cell cultures by RT-PCR as described [26 (link)] and analyzed using the IMGT/V-Quest program.

Cells were fixed and permeabilized with FIX & PERM Cell Permeabilization Kit (Invitrogen) and subsequently stained with CD45-KO (Beckman Coulter) and p-STAT1-PE or p-STAT3-PE (eBioscience). Samples were measured on the Beckman Coulter CytoFLEX. During analysis, cells were gated on being CD45⁺ to eliminate debris. Gating on monocyte and lymphocyte populations was done in the forward versus side scatter plot.