Scientists identify genes linked to high production of key antibody cnsiatucla NatureComms
Quantification of IgG signal on nanovials from cell secretions was performed through the following gating strategy illustrated in Supplementary Fig.. First, objects in focus were identified by the GradientRMS function in the brightfield channel. Single nanovial objects were separated from debris and multicellular aggregates based on an Aspect Ratio vs Area. Visual inspection of objects located in the gates drawn in S4B confirms the correct placement of gate settings.
Non-barcoded nanovial preparation . Nanovials were modified by mixing with a solution of 60 μg/mL streptavidin at equal volume following standard procedures as described above. The streptavidin-modified nanovial solution was then mixed with a solution containing an antibody cocktail comprising 8 μg/mL anti-mouse CD45 biotinylated using EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit and 20 μg/mL Goat anti-Mouse IgG FC at equal volume.
Raji and Hybridoma cells were loaded separately into nanovials on ice and incubated for 1 h to allow binding. Background cells were removed using a 20-μm cell strainer and cells were then incubated in a CO2 incubator at 37 °C for 30 min to accumulate secreted IgG. The hybridoma sample was stained with a 12 μg/mL solution of TotalSeqC conjugated anti IgG1 antibody and incubated for 45 min on Ice. Samples were then washed three times with ultra-pure PBS containing 0.04% BSA .
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