CAR moiety was labeled with a donkey anti-mouse Fab (Abcam, 150109). For T-cell phenotyping, the following antibodies were used: mouse anti-human PE-CD8 (Clone HIT8a, 12-0089-42), APC-CD56 (Clone TULY56, 17-0566-42) from eBiosciences; mouse anti-human APC-CD279 (Clone EH12.2H7, 329908), FITC-CD223 (Clone 11C3C65, 369308), Brilliant Violet 421-CD127 (Clone A019D5, 351310), PE/cy7-CD62L (Clone DREG-56, 304822), PE-biotin (Clone 1D4-C5, 409003) from Biolegend; and mouse anti-human Brilliant Violet 421-Tim-3 (Clone 7D3 (RUO), 565562) from BD Horizon. For intracellular staining, T cells were fixed and permeabilized using BD Cytofix/Cytoperm kit as the recommendation of the manufacturer. Anti-human CD8-PE (Clone HIT8a, eBiosciences, 12-0089-42) and donkey anti-mouse Fab (Polyclonal, Abcam, ab150109) were used for extracellular staining, and anti-human IFN-γ-eFluor 450 (Clone 4 S.B3, 85-48-7319-42, eBiosciences) was used for intracellular staining. Stained samples were acquired on a BD FACSAria and analyzed with FlowJo software (Tree Star, Ashland, OR).
Pe biotin
Manufactured by BioLegend
PE-biotin is a fluorescent conjugate used in flow cytometry. It consists of the fluorescent dye Phycoerythrin (PE) coupled to the small molecule biotin. This combination allows for the detection and labeling of target molecules that have an affinity for biotin.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria, by BD (1 mentions)
Cd56 apc, by Thermo Fisher Scientific (1 mentions)
Cd62l pe cy7, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Anti human cd8 pe, by Thermo Fisher Scientific (1 mentions)
Anti human ifn γ efluor 450, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Bovine serum albumin (bsa), by neoFroxx (1 mentions)
2 protocols using pe biotin
1
T-cell Immunophenotyping by Flow Cytometry
2
Biotin-CD28H Labeling of PBMCs
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In brief, PBMCs in each group were collected into a 15-ml centrifuge tube at 300 × g and 4°C for 10 min. The cell density was adjusted to 2×106/ml after discarding the supernatant. The cells were washed with PBS containing 2% bovine serum albumin (BSA; Biofroxx) 3 times, and then resuspended in 0.1 ml of ice-cold washing buffer and incubated with 1 µg biotin-CD28H [a kind gift from Professor Yuwen Zhu (Department of Surgery, University of Colorado Anschutz Medical Campus, Aurora, USA) (13 (link),15 (link))] for 45 min at 4°C. The cells were washed three times with 3 ml ice-cold washing buffer and then incubated with 1 µg PE-Biotin (cat. no. 409004; BioLegend, Inc.) for 30 min at 4°C. At the end of the incubation, the cells were washed three times with 3 ml ice-cold washing buffer and resuspended in 0.2 ml PBS for examination on a FACSCalibur flow cytometer (BD Biosciences).
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