Multiparametric Flow Cytometry Analysis

Blood drawn for the tail vein (50 μl) was collected with heparin-coated capillary tubes (Fisher Scientific, Pittsburgh, PA). Erythrocytes were lysed using ACK lysis buffer (Invitrogen, San Diego, CA) and white blood cells were analyzed by FACS using specific antibodies12 (link). Splenic and thymic cells were isolated as described1 (link)12 (link). Cell populations were quantified by FACS on mononuclear FSC/SSC gating using anti-human CD45 (clone #2D1), CD3 (#HIT3a), CD4 (#SK3), CD8 (#RPA-T8), and CD19 (#H1B19), HLA-DR (#tu39), HLA-A2 (#BB7.2), and intracellularly stained with granzyme B (#GB11) (BD Biosciences, San Diego, CA), perforin (#B-D48, BioLegend, San Diego, CA) or FOXP3 (#PCH101, eBiosiences, San Diego, CA) Abs. HLA-A2/GIL dextramers were obtained from Immudex (Copenhagen, Denmark) and GIL-specific CD8 T cells were enumerated by FACS following the manufacturer’s instructions.

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Majji S., Wijayalath W., Shashikumar S., Pow-Sang L., Villasante E., Brumeanu T.D, & Casares S. (2016). Differential effect of HLA class-I versus class-II transgenes on human T and B cell reconstitution and function in NRG mice. Scientific Reports, 6, 28093.

Publication 2016

heparin-coated capillary tubes ACK lysis buffer anti-human CD45 granzyme B

Blood Buffer Capillary Cd8 t cells Cell Clone Erythrocytes Granzyme b Heparin Hla a2 Hla dr Human Perforin Populations Splenic Tail Thymic Vein White blood cells

Corresponding Organization :

Other organizations : Walter Reed Army Institute of Research, Naval Medical Research Command, Uniformed Services University of the Health Sciences

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Blood drawn from the tail vein

dependent variables

Percentage of cell populations quantified by FACS
Enumeration of HLA-A2/GIL dextramers-specific CD8 T cells

control variables

Heparin-coated capillary tubes used for blood collection
ACK lysis buffer used for erythrocyte lysis
Specific antibodies used for FACS analysis
Splenic and thymic cells isolated as described in referenced studies

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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