BioLegend

Babco, Signet, Sternberger Monoclonals, Senetek, Covance

ELISA/assay

Rapid Human Treg ID Kit (FOXP3 Alexa Fluor® 488/CD4 APC/CD25 PE/CD3 PerCP/Cy5.5)

361801

25 tests

450 USD

FOXP3/CD4/CD25/CD3

Flow Cytometry, Intracellular Staining for Flow Cytometry

361801

206D

Rapid Human Treg ID Kit (FOXP3 Alexa Fluor® 488/CD4 APC/CD25 PE/CD3 PerCP/Cy5.5)

Forkhead box protein P3, Scurfin, JM2, IPEX, Zinc finger protein JM2

25 tests

450 USD

Human

Monoclonal

Materials provided: 1. Anti-human FOXP3 Alexa Fluor 488/CD25 PE/CD3 PerCP/Cy5.5/CD4 APC antibody cocktail, 25 tests 2. Mouse IgG1, isotype control Alexa Fluor 488/CD25 PE/CD3 PerCP/Cy5.5/CD4 APC antibody cocktail, 25 tests 3. Buffer 1, Buffer 2, and Buffer 3, 50 tests ( Dilute Buffer 3 by mixing one part 10X Buffer 3 with 9 parts of PBS. ) Materials not provided: 1. 1X PBS 2. Fetal bovine serum 3. Single color compensation controls Protocol for staining whole blood: a) Add 50 uL of blood, followed by 5 ul of Buffer 1, to the bottom of each appropriately labeled tube. b) Immediately mix by vortexing, and incubate at room temperature for 15 minutes. c) Vortex, add 300 uL of the Buffer 2, and vortex again. d) Add 20 ul of the appropriate (isotype or FOXP3) cocktail to the indicated tube. e) Vortex and incubate at room temperature, in the dark, for 60 minutes. f) Add 3 mL PBS, mix and incubate for 5 minutes. Centrifuge at 500x g for 5 min. Discard the supernatant. g) Add 3 mL of 1X Buffer 3 (prepared from the 10X concentrated Buffer 3 solution); vortex and centrifuge at 500g for 5 min. Discard the supernatant. h) Resuspend the cell pellet in 500 uL of 1X Buffer 3 and acquire on a cytometer equipped with appropriate lasers. Protocol for staining PBMCs: a) Resuspend PBMCs in 100% serum (FCS or FBS), at a concentration between 5x10 6 /ml and 4x10 7 /ml. b) Pipet 50 ul of the cell suspension to the bottom of appropriately labeled tubes, then add 2.5 ul of Buffer 1 to each tube. c) Vortex and incubate at room temperature for 15 minutes. d) Add 150 ul of Buffer 2, folllowed by 20 ul of the appropriate (isotype or FOXP3) cocktail. e) Vortex and incubate at room temperature, in the dark, for 60 minutes. f) Add 1.5 to 3.0 ml of 1X Buffer 3 (prepared by diluting 10X concentrated Buffer 3 solution), vortex, and centrifuge at 500x g for 5 minutes. Discard supernatant. g) Resuspend the cells in 500 ul of 1X Buffer 3 reagent and acquire on a cytometer equipped with the appropriate lasers. For data analysis, it is recommended to create a SSC vs CD3 plot, gating on CD3+ cells. FOXP3+ CD25+ T cells can then be identified by gating on CD3+ CD4+ cells.