Polycarbonate 96 well transwell plates

Chemotaxis assays were performed as previously described [3 (link)]. Briefly, RAMOS human B cells were resuspended in freshly prepared migration media (RPMI + 1% BSA + 100 U/mL Pen/Strep), incubated for 1 h, and $5\times {10}^5$ cells (in 75 µL ) loaded into the upper chambers of 5 µm polycarbonate 96-well transwell plates (Corning, Corning, NY, USA). In triplicate wells, migration media containing varying concentrations of CXCL12 (0 ng/mL to 500 ng/mL) were added to the bottom chambers of the transwells (235 µL per well). Plates were incubated for 1 h at varying O ${}_2$ levels as described above. Upper transwells were removed and 100 µL of Cell Titer Glo (Promega, Madison, WI, USA) added to each bottom well; plates were then placed on an orbital shaker for 2 min and incubated at RT for 10 min in the dark. Luminescence was measured on a SynergyTM HT microplate reader (BioTek Instruments, Winooski, VT, USA) and relative luminescent units (RLU) reported.

The human B cell line, RAMOS cells, or magnetic bead enriched primary human peripheral blood or mouse splenic B cells were resuspended in freshly prepared migration media (PBS + 1% BSA), and 5 x 10⁵ cells (in 75 μL) loaded into the upper chambers of 5 μm polycarbonate 96 well transwell plates (Corning, Corning, NY). In triplicate wells, migration media containing varying concentrations of CXCL12 or CXCL13 was added to the bottom chambers of the transwells (225 μL per well). Plates were incubated for 1 hour at appropriate O₂ levels in the C-chamber incubator inserts (Biospherix, Parish, NY), at 37^∘C and 5% CO₂. Upper transwells were removed and 100 μL of Cell Titer Glo (Promega, Madison, WI) added to each bottom well and incubated at RT for 10 min. Luminescence was readout on a SynergyTM HT microplate reader (BioTek Instruments, Winooski, VT) and relative luminescent units (RLU) reported. Supplementary Figure 4 demonstrates the direct correlation between RLU and cell numbers, through a 4- log₁₀ range, independent of O₂ levels.