Phenotypic Analysis of Leukocytes

For phenotypical analysis of peripheral leukocytes, spleen samples were dissolved to single cell suspension, erythrocytes were lysed and cell numbers were determined as described previously^{25 (link)}. After blocking the FC-receptor II/III by preincubation with a CD16/CD32-specific Ab (clone 2.4G2; BioXCell), dead cells were stained using the LIVE/DEAD® fixable dead cell stain kit (Invitrogen^TM GmbH). Subsequently, CD4- (PacificBlue; clone GK1.5; BioLegend), CD8α- (HV500; clone 53-6.7; BD Biosciences), CD19- (PE-eFluor610; clone 1D3; eBioscience/Thermo Fisher Scientific), CD69- (FITC; clone H1.2F3; BioLegend) and CD44-specific Ab (APC; clone IM7; BioLegend) were added for fluorochrome-conjugated surface marker staining. For intracellular staining of Foxp3 the Foxp3 Transcription Factor Staining Buffer Set (eBioscience/Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Samples were acquired with an LSRII SORP cytometer (BD Biosciences) and analyzed using FlowJo software version 9.6.4 (Tree Star, Ashland, USA). Blood samples were stained according to the same procedure.

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Uhde A.K., Ciurkiewicz M., Herder V., Khan M.A., Hensel N., Claus P., Beckstette M., Teich R., Floess S., Baumgärtner W., Jung K., Huehn J, & Beineke A. (2018). Intact interleukin-10 receptor signaling protects from hippocampal damage elicited by experimental neurotropic virus infection of SJL mice. Scientific Reports, 8, 6106.

Publication 2018

LIVE/DEAD® fixable dead cell stain kit CD4- (PacificBlue; clone GK1.5; Foxp3 Transcription Factor Staining Buffer Set LSRII SORP cytometer

Blood Buffer Cd44 Cell Clone Erythrocytes Fc receptor Fitc Fluorochrome Intracellular Leukocytes analysis Spleen Transcription factor Tree

Corresponding Organization :

Other organizations : University of Veterinary Medicine Hannover, Foundation, Medizinische Hochschule Hannover, Helmholtz Centre for Infection Research

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Phenotypical analysis of peripheral leukocytes
Proportion of CD4+, CD8+, CD19+, CD69+, CD44+ cells
Foxp3 expression

control variables

Spleen samples were dissolved to single cell suspension
Erythrocytes were lysed
Cell numbers were determined as described previously
FC-receptor II/III was blocked by preincubation with a CD16/CD32-specific Ab
Dead cells were stained using the LIVE/DEAD® fixable dead cell stain kit
Fluorochrome-conjugated surface markers (CD4, CD8α, CD19, CD69, CD44) were used for staining
Foxp3 was stained using the Foxp3 Transcription Factor Staining Buffer Set
Samples were acquired with an LSRII SORP cytometer and analyzed using FlowJo software
Blood samples were stained according to the same procedure

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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