Detailed Flow Cytometry Staining Protocol

Abs used in flow cytometric analyses were as described in Lang et al. (2011 (link), 2013 (link)). Abs specific for mGR1 (RB6-8C5), mH-2D^d (34-2-12), mCD45 (30-F11), hCD3 (OKT3, HIT3a), hCD5 (UCHT1), hCD34 (581), hCD38 (HB-7), hCD40 (5C3), hCD45 (H130), hCD138 (Ml15), hCD268 (11C1), hIgκ (MHK-49), hIgλ (MHL-38), and hIgM (MHM-88) were from BioLegend. Abs for rat IgG₁ (RG11/39.4), hCD3 (HIT3a), hCD19 (HIB19), and hIgM (G20-127) were from BD, and those for hIgκ (HP6062) and hIgM (SA-DA4) were from eBioscience. Fab′ goat anti-hIgM was from Protos Immunoresearch. Cells were stained in staining buffer (PBS, 1% BSA, 0.1% Na Azide) for 15 min at 4°C and washed twice with the same buffer. Samples were collected on CyAn analyzers (Beckman Coulter) at the flow cytometry cores at NJH or UCD Cancer Center, and analyses were performed with FlowJo software as described in Lang et al. (2013) (link).

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Lang J., Ota T., Kelly M., Strauch P., Freed B.M., Torres R.M., Nemazee D, & Pelanda R. (2016). Receptor editing and genetic variability in human autoreactive B cells. The Journal of Experimental Medicine, 213(1), 93-108.

Publication 2016

RB6-8C5), MHM-88)

Azide Buffer Cancer Cells Flow cytometry Goat Higm Igg1 Okt3

Corresponding Organization : National Jewish Health

Other organizations : Scripps Research Institute

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Variable analysis

independent variables

Antibodies (Abs) specific for mGR1 (RB6-8C5), mH-2Dd (34-2-12), mCD45 (30-F11), hCD3 (OKT3, HIT3a), hCD5 (UCHT1), hCD34 (581), hCD38 (HB-7), hCD40 (5C3), hCD45 (H130), hCD138 (Ml15), hCD268 (11C1), hIgκ (MHK-49), hIgλ (MHL-38), and hIgM (MHM-88)

dependent variables

Cell staining characteristics measured by flow cytometry

control variables

Staining buffer (PBS, 1% BSA, 0.1% Na Azide)
Staining conditions (15 min at 4°C)
Flow cytometry platforms (CyAn analyzers from Beckman Coulter)
Analysis software (FlowJo)

positive controls

Rat IgG1 (RG11/39.4)
HCD3 (HIT3a)
HCD19 (HIB19)
HIgM (G20-127)

negative controls

Fab' goat anti-hIgM (from Protos Immunoresearch)

Annotations

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