Multiparametric Flow Cytometry Staining

Staining for surface markers was performed by incubating cells with antibody at 1:200 dilutions in fluorescence-activated cell sorting buffer (0.1% BSA in PBS) for 30 min at 4 °C. For intracellular cytokine staining of IFNγ and TNFα, surface markers were stained before fixation/permeabilization (BD Cytofix/Cytoperm Kit, #554714; BD Biosciences) (40 (link)). Flow antibodies were purchased from BioLegend with the information as follows: CD8a-BV786 (#563332), CD11c-BV786 (#563735), PD1-PE/Cy7 (#109109), TIM3-APC (#119705), KLRG1-FITC (#138409), LAG3-PerCP/Cy5.5 (#125211), TNFα-PE/Cy7 (#506323), and Granzyme B-Alexa 700 (#372221). Samples were acquired on BD LSRFortessa flow cytometer and analyzed with FlowJo software (https://www.flowjo.com/solutions/flowjo) (Tree Star) unless otherwise stated.

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Zhang J., Ye Z.W., Bräutigam L., Chakraborty P., Luo Z., Culpepper J., Aslam M., Zhang L., Johansson K., Haeggström J.Z., Xu J., Olsson M., Townsend D.M., Mehrotra S., Morgenstern R, & Tew K.D. (2023). A role for microsomal glutathione transferase 1 in melanin biosynthesis and melanoma progression. The Journal of Biological Chemistry, 299(8), 104920.

Publication 2023

BD Cytofix/Cytoperm Kit CD11c-BV786 PD1-PE/Cy7 TIM3-APC KLRG1-FITC LAG3-PerCP/Cy5.5 TNFα-PE/Cy7 BD LSRFortessa flow cytometer

Antibodies Antibody Buffer Cells Cy5 5 Cytokine Dilutions Fitc Granzyme b Ifnγ Intracellular Klrg1 Tim3 Tnfα Tree

Corresponding Organization : Medical University of South Carolina

Other organizations : Karolinska Institutet, Pfizer (Sweden), Dalian University of Technology

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Variable analysis

independent variables

Antibody dilution (1:200)
Incubation time (30 min)
Incubation temperature (4 °C)

dependent variables

Expression of surface markers (CD8a, CD11c, PD1, TIM3, KLRG1, LAG3)
Expression of intracellular cytokines (IFNγ, TNFα)
Expression of Granzyme B

control variables

Fluorescence-activated cell sorting (FACS) buffer composition (0.1% BSA in PBS)
Fixation/permeabilization using BD Cytofix/Cytoperm Kit (#554714)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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