TABLE 3

Antibodies used for flow cytometryc

ManufacturerEpitopeCloneFluorochrome for conjugationConcn of antibody used per test (ng)
BD PharmingenTCR Vβ4KT4PE500
TCR Vβ5.1 5.2MR9-4FITC400
TCR Vβ8F23.1FITC1,500
TCR Vβ8F23.1PE500
TCR Vβ1414-2FITC100
BioLegendB220RA3-6B2FITC50a
CD317A2FITC50a
CD3ε145-2C11APC125
CD4RM4-5PE-Cy7125
CD8a53-6.7Biotin3.125 per million cells (for depletion)
CD8a53-6.7FITC50a (for lineage stain)
1,000 (for single stain)
CD8a53-6.7APC250
CD8a53-6.7Brilliant Violet 51025 per million cells
CD11cN418FITC12.5a
CD196D5FITC12.5a
CD25PC61PE-Cy725
CD44IM7PerCP-Cy5.550
CD62LMEL-14APC25
CD117 (cKit)2B8APC25
CD117 (cKit)2B8APC-Cy725
GATA316E10A23PE0.75 per million cells
Gr1RB6-8C5FITC12.5a
Mac1M1/70FITC12.5a
NK1.1PK136FITC12.5a
TER119TER-119FITC50a
TCRβH57-597FITC12.5a
TCR Vα2B20.1FITC50
TCR Vα3.2RR3-16PE50
TCR Vα8.3B21.14PE250
TCR Vα11.1 11.2RR8-1PE300
TCR Vβ2B20.6PE2,000
TCR Vβ5.1 5.2MR9-4PE125
TCR Vβ6RR4-7PE50
TCR Vβ7TR310PE125
TCR Vβ9MR10-2PE25
TCR Vβ11RR3-15PE100
TCR Vβ12MR11-1PE60
TCR Vβ13MR12-4PE200
TCRγδGL3FITC12.5a
Thy1.253-2.1PerCP-Cy5.5100
eBioscienceB220RA3-6B2eFluor 45050b
CD317A2eFluor 45050b
CD3ε145-2C11eFluor 45060
CD8a53-6.7eFluor 45012.5b (for lineage stain)
CD11cN418eFluor 45012.5b
CD19eBio1D3eFluor 45012.5b
CD25PC61.5PerCP-Cy5.550
CD27LG.7F9APC25
Gr1RB6-8C5eFluor 45012.5b
Mac1M1/70eFluor 45012.5b
NK1.1PK136eFluor 45012.5b
TER119TER-119eFluor 45050b
TCRβH57-597eFluor 45012.5b
TCR Vβ10bB21.5PE100
TCRγδeBioGL3eFluor 45012.5b
  • a These antibody cocktails were used to exclude mature hematopoietic-lineage cells in thymocytes by conjugated fluorochrome FITC.

  • b These antibody cocktails were used to exclude mature hematopoietic-lineage cells in thymocytes by conjugated fluorochrome eFluor 450.

  • c Antibodies were titrated at six concentrations to determine the optimized usage prior to these experiments. Each test can be applied to as many as 15 × 106 cells in a 50- to 100-μl volume, unless otherwise noted. Isotype controls (if used) were used at the same amounts as the corresponding experimental antibody. PE, phycoerythrin; FITC, fluorescein isothiocyanate; APC, allophycocyanin; PerCP, peridinin chlorophyll protein.