The phenotype of expanded TIL was evaluated using flow cytometry. The antibody panel used included CD3 PerCpCy5.5 (Biolegend 300430), CD4 FITC (BD Biosciences 555346), CD8 BV510 (Biolegend 344732), and CD56 PE (BD Biosciences 555516). All cells were stained with a Live/Dead Near-IR viability stain (Invitrogen, L10119) and fixed in 2% paraformaldehyde. Data were acquired on an LSR II or Celesta flow cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar, Inc.). Cells expressing CD56 without CD3 (CD3−CD56+) were labelled NK cells whereas cells expressing T cell markers of CD3, CD4 and CD8 were considered CD3+ T cells, CD4+ T cells and CD8+ T cells respectively.
Live dead near ir viability stain
Manufactured by Thermo Fisher Scientific
The Live/Dead Near-IR viability stain is a fluorescent dye that can be used to distinguish between live and dead cells in a sample. The stain selectively labels dead cells, allowing for their identification and quantification using near-infrared fluorescence microscopy or flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii flow cytometer, by Tree Star (2 mentions)
Cd3 percp cy5, by BioLegend (1 mentions)
Cd8 bv510, by BioLegend (1 mentions)
Cd56 pe, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Celesta flow cytometer, by BD (1 mentions)
Cd11b, by BD (1 mentions)
Human ifng quantikine elisa kit, by R&D Systems (1 mentions)
3 protocols using live dead near ir viability stain
1
Expanded TIL Phenotypic Characterization
2
Immunophenotyping of Tumor-Infiltrating Lymphocytes
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Digested tumors were stained with fluorescent antibodies including CD3 (BD Biosciences, BDB563546), CD4 (BD Biosciences, BDB563875), CD8 (Biolegend, 300928), foxp3 (BD Biosciences, BDB560046), CD19 (BD Biosciences, BDB560728), and CD11b (BD Biosciences, BDB550019) to identify immune cell subsets. Expanded TIL was stained with fluorescent antibodies for CD3, CD4, CD8, and CD56 (BD Biosciences, BDB555516). All cells were stained with a Live/Dead Near-IR viability stain (Invitrogen, L10119) and fixed in 2% paraformaldehyde. Data were acquired on an LSR II flow cytometer and analyzed using FlowJo software (Treestar, Inc.).
3
TIL Characterization and Tumor Reactivity
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Flow cytometric analysis of TIL from each fragment was performed. Expanded TIL was stained with fluorescent antibodies for CD3, CD4, CD8, and CD56 (BD Biosciences, BDB555516). All cells were stained with a Live/Dead Near-IR viability stain (Invitrogen, L10119) and fixed in 2% paraformaldehyde. Data were acquired on an LSR II flow cytometer and analyzed using FlowJo software (TreeStar, Inc.). TIL and autologous tumor cells from enzymatic digestion were cultured at a 1:1 ratio (1 x 105 cells each) overnight in round bottom 96-well plates. Supernatants were collected after 24 h. IFN-gamma was measured using a Human IFNg Quantikine ELISA Kit (R&D Systems, SIF50). Optical density of each well was measured at 450 nm and IFN-γ concentration was calculated from the standard curve. IFN-γ concentration ≥100 pg/ml was the cut-off for reactivity.
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