Streptomycin sulfate

Limiting dilution assays were performed as previously described [18 (link)]. Briefly, peritoneal exudate cells (PECs) were collected by lavage in 10 ml of complete D10 (DMEM (Corning), 10% FBS (Biowest), 2mM L-glutamine (Gibco), 1% HEPES (Corning), 10 U/mL Penicillin and 10 μg/mL streptomycin sulfate (Corning)) and washed once. Cells were counted. In addition, spleens were collected and homogenized in glass tissue disrupters. Splenocytes were RBC lysed (ACK lysing buffer, Gibco), washed, and counted. 2 x 10⁶ PECs or 5 x 10⁶ splenocytes were used for the 0 dilution. Two-fold dilutions were performed and plated on C57BL/6 mouse embryonic fibroblasts. For disrupted samples, 2 x 10⁶ PECs or 5 x 10⁶ splenocytes were homogenized in a Precellys 24 (Bertin Instruments) at 6000 rpm for 1 min. Two-fold dilutions were plated of the disrupted samples. The assay was scored for cytopathic effect three weeks after plating.

PECs were harvested by peritoneal lavage with 10 ml of complete D5 ((DMEM (Corning), 5% FBS (Biowest), 2mM L-glutamine (Gibco), 1% HEPES (Corning), 10 U/mL Penicillin and 10 μg/mL streptomycin sulfate (Corning)) and washed once. The PECs were resuspended in 1 ml of D5, then were homogenized with 1 mm zirconia beads (BioSpec Products) in a Precellys 24 (Bertin Instruments) at 5000 rpm for 1 min. 10-fold serial dilutions were plated on 3T12s. Virus was absorbed for 1 hour at 37 degrees C before 1% (w/v) methylcellulose (Sigma-Aldrich) (10 g methylcellulose in 1L MEM (Corning)) was added as an overlay. Plates were incubated at 37 degrees C in 5% CO₂ for 7 days before fixing with 2% formaldehyde and staining with 0.1% crystal violet (Sigma-Aldrich).

Compounds, SDF-1α (PeproTech; 30028A), and DMSO (Sigma Aldrich; St. Louis, MO, USA) normalization to 0.5% were dispensed using the TECAN D300e digital dispenser into a 384-well black wall clear-bottom microplate (Corning; Corning, NY, USA). Tango™ CXCR4-bla U2OS Cells were resuspended in assay media consisting of DMEM (Corning; Corning, NY, USA) supplemented with 1% dialyzed Fetal bovine serum, 0.1 mM MEM Non-essential amino acids solution, 25 mM HEPES solution (Corning; Corning, NY, USA), 1 mM Sodium Pyruvate and 100 IU penicillin, 100 mg/mL streptomycin sulfate (Corning; Corning, NY, USA) at a density of 3.0 × 10⁵ cells/mL. Cells suspension was dispensed at 30 μL per well into assay plate and incubated overnight in a cell culture incubator at 37 °C with 5% CO₂. Following overnight incubation, LiveBlazer FRET B/G Loading Kit working reagent was prepared according to supplier guidelines and 6 μL of solution was dispensed into each well. Assay plate was covered and incubated in a dark place for two hours and Fluorescence Intensity was measured using the BMG LABTECH CLARIOstar Plus (BMG LABTECH Inc., Cary, NC, USA) according to the LiveBlazer FRET B/G Loading Kit excitation and emission measurement guidelines.