Individual or custom antibodies can be purchased through BioLegend to create custom antibody panels that can be used alone or in conjunction with the pre-titrated TotalSeq™-D Heme Oncology Cocktail.
Briefly, custom antibody cocktails are made by mixing solubilized antibodies with Cell Staining Buffer (CSB) to reach a total volume of 60 μL. This 60 μL cocktail can be used alone (spin 60 μL cocktail for 15 min, 14,000 x g, 4 °C and use 50 μL for staining) or used in lieu of CSB to reconstitute the lyophilized TotalSeq™-D Heme Oncology Cocktail as outlined in the DNA + Protein User Guide.
Custom antibodies are supplied in solution at 0.5 µg/µL. We recommend reaching out to BioLegend support to request titration values for each custom antibody. This will help in preparation of the cocktail using the spreadsheet below. If BioLegend does not have specific titration values, we recommend starting with 0.5 μg (1 μL) per antibody for an initial Tapestri run.
Hashtag use with custom protein panels
For hashing with custom protein panels of any size, the suggested starting hashtag amount is 0.05 ug per 500,000 to 1 million cells in a 100 µL volume. For a 0.05 ug input, you may choose to make serial dilutions. Hashtag concentrations may need to be adjusted to improve performance. It is recommended to increase the hashtag amount if the percentage of unassigned cells to sample is > 30%, or decrease the hashtag amount if the fraction of reads to hashtag(s) is too high.