Multi-Color Flow Cytometry of Lung CD8+ T Cells

Single-cell suspensions were prepared from lung tissue as described (22 (link), 48 (link)). Unspecific staining was blocked with unconjugated anti-FcγRII/III antibody (anti-CD16/CD32; clone 2.4G2, BD Pharmingen, Heidelberg, Germany). Cells were specifically stained with the following antibodies for multi-color cytofluorometric analyses: ECD-conjugated anti-CD8α (clone 53-6.7; Beckman Coulter, Krefeld, Germany), FITC-conjugated anti-KLRG1 (clone 2F1; eBioscience, Frankfurt), PE-Cy5-conjugated anti-CD127 (clone A7R34; eBioscience, Frankfurt), and PE-Cy7-conjugated anti-CD62L (clone MEL-14; Beckman Coulter). Phenotypic characterization of peptide-specific CD8⁺ T cells was performed using PE-conjugated dextramers H-2Ld/YPHFMPTNL (IE1), H-2Dd/AGPPRYSRI (m164), and H-2Kd/TYWPVVSDI (M105) (22 (link), 31 (link)). H-2Kb/SIINFEKL served as the control for excluding unspecific staining (Immudex, Copenhagen, Denmark). For the analyses, a “live gate” was routinely set on leukocytes in the forward scatter (FSC) versus sideward scatter (SSC) plot. All cytofluorometric analyses were performed with flow cytometer FC500 and CXP analysis software (Beckman Coulter).

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Griessl M., Renzaho A., Freitag K., Seckert C.K., Reddehase M.J, & Lemmermann N.A. (2021). Stochastic Episodes of Latent Cytomegalovirus Transcription Drive CD8 T-Cell “Memory Inflation” and Avoid Immune Evasion. Frontiers in Immunology, 12, 668885.

Publication 2021

anti-CD16/CD32; clone 2.4G2, ECD-conjugated anti-CD8α (clone 53-6.7; FITC-conjugated anti-KLRG1 (clone 2F1; PE-Cy5-conjugated anti-CD127 (clone A7R34; PE-Cy7-conjugated anti-CD62L (clone MEL-14; flow cytometer FC500 CXP analysis software

Antibodies Cd62l Cd8 t cells Cells Clone Fcγrii Fitc Klrg1 Leukocytes Lung Peptide Phenotypic Tissue

Corresponding Organization : University Medical Center of the Johannes Gutenberg University Mainz

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Variable analysis

independent variables

Preparation of single-cell suspensions from lung tissue

dependent variables

Phenotypic characterization of peptide-specific CD8+ T cells

control variables

Unspecific staining was blocked with unconjugated anti-FcγRII/III antibody (anti-CD16/CD32; clone 2.4G2, BD Pharmingen, Heidelberg, Germany)
H-2Kb/SIINFEKL served as the control for excluding unspecific staining (Immudex, Copenhagen, Denmark)

positive controls

H-2Ld/YPHFMPTNL (IE1)
H-2Dd/AGPPRYSRI (m164)
H-2Kd/TYWPVVSDI (M105)

negative controls

H-2Kb/SIINFEKL

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