A CD45-based barcoding approach to multiplex mass-cytometry (CyTOF).

Cytometry. Part A : the journal of the International Society for Analytical Cytology (2015-02-04)
Liyun Lai, Raymond Ong, Juntao Li, Salvatore Albani

CyTOF enables the study of the immune system with a complexity, depth, and multidimensionality never achieved before. However, the full potential of using CyTOF can be limited by scarce cell samples. Barcoding strategies developed based on direct labeling of cells using maleimido-monoamide-DOTA (m-DOTA) provide a very useful tool. However, using m-DOTA has some inherent problems, mainly associated with signal intensity. This may be a source of uncertainty when samples are multiplexed. As an alternative or complementary approach to m-DOTA, conjugating an antibody, specific for a membrane protein present on most immune cells, with different isotopes could address the issues of stability and signal intensity needed for effective barcoding. We chose for this purpose CD45, and designed experiments to address different types of cultures and the ability to detect extra- and intra-cellular targets. We show here that our approach provides an useful alternative to m-DOTA in terms of sensitivity, specificity, flexibility, and user-friendliness. Our manuscript provides details to effectively barcode immune cells, overcoming limitations in current technology and enabling the use of CyTOF with scarce samples (for instance precious clinical samples).

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Phorbol 12-myristate 13-acetate, ≥99% (TLC), film or powder
Ionomycin calcium salt from Streptomyces conglobatus, powder, ≥98% (HPLC)
Brefeldin A, from Penicillium brefeldianum, ≥99% (HPLC and TLC)
Brefeldin A, ≥99% (HPLC and TLC), BioXtra, for molecular biology
Phorbol 12-myristate 13-acetate, synthetic, ≥98.0% (TLC)
Brefeldin A, from Penicillium brefeldianum, Ready Made Solution, 10 mg/mL in DMSO