Multiparametric Flow Cytometry of Lung Immune Cells

Single cell suspensions were prepared from isolated lungs and stained for flow cytometry as described for nasal epithelial tissue [21 (link)]. Following fluorescent-labeled antibodies were used: APC/cy7*anti-rat CD45 (#202216, BD Biosciences), Brilliant Violet 421*anti-rat CD3 clone 1F4 (RV0) (#563948, BD Biosciences), APC*anti-rat CD11b clone WTS (RV0) (#562102 BD Biosciences), FITC* anti-rat RT1Dab (MHCII) (#205405, BioLegend, Inc.), PerCP/cy 5.5* anti-rat CD4 (#201519, BioLegend, Inc.), and PE* anti-rat CD161 (#205604, BioLegend Inc.). Live lymphocyte sized cells were stained with CD45 and CD3 to identify T cells. Further, the T cells were classified into T helper and cytotoxic T cells based on CD4 and CD8 expression, respectively. CD3-, CD11b-, CD4+ and MHCII+ cells were identified as plasmacytoid dendritic cells (pDCs). CD3- and CD161+ cells were identified as natural killer (NK) cells. Gating strategies have been elaborated in detail in our previous publication [21 (link)].

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Giri T., Panda S, & Palanisamy A. (2023). Pregnancy-induced differential expression of SARS-CoV-2 and influenza a viral entry factors in the lower respiratory tract. PLOS ONE, 18(7), e0281033.

Publication 2023

APC/cy7*anti-rat CD45 Brilliant Violet 421*anti-rat CD3 clone 1F4 (RV0) APC*anti-rat CD11b clone WTS (RV0) FITC* anti-rat RT1Dab (MHCII) PerCP/cy 5.5* anti-rat CD4

Anti cd3 Cd11b Cd161 Cells Clone Cytotoxic t cells Fitc Flow cytometry Fluorescent antibodies Lungs Lymphocyte Nasal epithelial tissue Natural killer cells Plasmacytoid dendritic cells T cells Violet

Corresponding Organization : Washington University in St. Louis

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Variable analysis

independent variables

Isolated lungs

dependent variables

Percentage of T cells (CD45+, CD3+)
Percentage of T helper cells (CD4+)
Percentage of cytotoxic T cells (CD8+)
Percentage of plasmacytoid dendritic cells (CD3-, CD11b-, CD4+, MHCII+)
Percentage of natural killer cells (CD3-, CD161+)

control variables

Preparation of single cell suspensions from isolated lungs
Staining procedure for flow cytometry, as described for nasal epithelial tissue in the referenced publication [21]
Fluorescent-labeled antibodies used for staining: APC/cy7*anti-rat CD45, Brilliant Violet 421*anti-rat CD3, APC*anti-rat CD11b, FITC* anti-rat RT1Dab (MHCII), PerCP/cy 5.5* anti-rat CD4, and PE* anti-rat CD161

controls

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

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