Multiparametric Flow Cytometry for Immune Cell Profiling

Single-cell suspensions of in vitro generated DCs or murine tumors, spleens and lymph nodes were incubated with antibodies for 20 min at 4°C, followed by washing with staining buffer (DPBS+2% FBS). Surface staining and intracellular staining for FOXP3 and Ki-67 were performed as previously described.26 (link) IFNγ production was evaluated by intracellular staining after in vitro stimulation with Cell Stimulation Cocktail (eBioscience) for 4 hours, using the intracellular fixation and permeabilization buffer set (eBioscience). Data acquisition was performed on Attune NxT (ThermoFisher) or NovoCyte Quanteon (Agilent) cytometer, and data analyzed by FlowJo software (TreeStar). Antibodies are detailed in online supplemental table S1.

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Salehi-Rad R., Lim R.J., Du Y., Tran L.M., Li R., Ong S.L., Ling Huang Z., Dumitras C., Zhang T., Park S.J., Crosson W., Kahangi B., Abascal J., Seet C., Oh M., Shabihkhani M., Paul M., Krysan K., Lisberg A.E., Garon E.B., Liu B, & Dubinett S.M. (2023). CCL21-DC in situ vaccination in murine NSCLC overcomes resistance to immunotherapy and generates systemic tumor-specific immunity. Journal for Immunotherapy of Cancer, 11(9), e006896.

Publication 2023

Cell Stimulation Cocktail intracellular fixation and permeabilization buffer set Attune NxT NovoCyte Quanteon

Antibodies Buffer Cell Ifnγ Intracellular Lymph nodes Murine Tumors

Corresponding Organization : University of California, Los Angeles

Other organizations : Zhejiang University

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Variable analysis

independent variables

In vitro generated DCs or murine tumors, spleens and lymph nodes

dependent variables

IFNγ production
Surface marker expression
FOXP3 and Ki-67 expression

control variables

Incubation time (20 min)
Incubation temperature (4°C)
Staining buffer (DPBS+2% FBS)
Intracellular fixation and permeabilization buffer set (eBioscience)
Cell Stimulation Cocktail (eBioscience) for 4 hours

controls

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

Annotations

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