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. 2022 May;101(5):374-379.
doi: 10.1002/cyto.a.24545. Epub 2022 Mar 10.

OMIP 083: A 21-marker 18-color flow cytometry panel for in-depth phenotyping of human peripheral monocytes

Kathryn E Hally  1   2   3 , Laura Ferrer-Font  4 , Katherine R Pilkington  5 , Peter D Larsen  1   2   3
Affiliations

OMIP 083: A 21-marker 18-color flow cytometry panel for in-depth phenotyping of human peripheral monocytes

Kathryn E Hally et al. Cytometry A. 2022 May.
No abstract available

Keywords: OMIP; PBMCs; full spectrum flow cytometry; human immunophenotyping; innate immunity; monocytes.

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Conflict of interest statement

Katherine Pilkington is an employee of Cytek Biosciences, Inc., the manufacturer of the Aurora full spectrum flow cytometer used in this manuscript.

Figures

FIGURE 1
FIGURE 1
Overview of the 21‐marker, 18‐color monocyte‐centric panel on human PBMCs. PBMCs were density‐separated from Cyto‐Chex blood collection tubes (Streck, La Vista, NE), stained and acquired on a 3‐laser Cytek Aurora (Cytek biosciences, Fremont, CA). Initially, the sample is cleaned by excluding variable flow rate during acquisition (typically experienced at the start and end of sample acquisition), doublets, debris and antibody aggregates (A, i‐vi). Following cleaning, CD45+ (A, vii) and CD66b− (A, viii) leukocytes are sequentially gated. Several lineage markers (CD3, CD19, CD56, CD123) are conjugated to the same fluorochrome to facilitate efficient exclusion of all other nonmonocyte cells (A, ix). Within the same plot that Lin+ leukocytes are excluded, the monocyte population is defined as TLR2+ (A, ix). As such, the monocyte population is defined as: CD45+/CD66b−/Lin−/TLR2+. Monocytes display their characteristic expression of CD14 and CD16 (B, i). Separating classical (CD14++/CD16−) monocytes from all other monocyte subsets (CD14var/CD16var) can be achieved in this plot. From here, classical monocytes are confirmed to display their expected pattern of CCR2 and CX3CR1 (B, ii). Residual classical monocytes are defined in B, iii as those cells which were originally gated as ‘other monocyte subsets’ from the parent population but which subsequently show CCR2 and CX3CR1 expression patterns identical to classical monocytes. This population is removed from downstream analysis. From here, a true population of ‘other monocyte subsets’ is defined (B, iii) and subsequently divided into intermediate (CCR2+/SLAN−) monocytes and two subsets of nonclassical monocytes based on expression of SLAN: SLAN+ (CCR2−/SLAN+) nonclassical and SLAN− (CCR2−/SLAN−) monocytes (B, iv). High‐dimensional analysis with the UMAP algorithm was conducted on PBMCs from two individuals across three timepoints (for a total of six samples) (C). Data were cleaned (as demonstrated in A, i‐vi) and CD45+ events from these six samples were combined and used for this analysis. Manual gating of the four monocyte populations was overlaid on this two‐dimensional rendering (C, i). UMAP analysis demonstrates the ease of discriminating the monocyte population from all other leukocytes using TLR2 (C, ii). Heatmap overlays also demonstrate the distinct expression levels of CD14 (C, iii), CD16 (C, iv), CCR2 (C, v), CX3CR1 (C, vi) and SLAN (C, vii) within these monocyte populations. Once identified, these four monocyte subsets were interrogated for their expression of a further nine markers (D); each monocyte subset shows distinctive expression profiles
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References

    1. Park LM, Lannigan J, Jaimes MC. OMIP‐069: forty‐color full Spectrum flow cytometry panel for deep Immunophenotyping of major cell subsets in human peripheral blood. Cytometry A. 2020;97(10):1044–51. - PMC - PubMed
    1. Niewold P, Ashhurst TM, Smith AL, King NJC. Evaluating spectral cytometry for immune profiling in viral disease. Cytometry A. 2020;97(11):1165–79. - PubMed
    1. Bocsi J, Melzer S, Dähnert I, Tárnok A. OMIP‐023: 10‐color, 13 antibody panel for in‐depth phenotyping of human peripheral blood leukocytes. Cytometry A. 2014;85(9):781–4. - PubMed
    1. Moncunill G, Han H, Dobaño C, McElrath MJ, De Rosa SC. OMIP‐024: pan‐leukocyte immunophenotypic characterization of PBMC subsets in human samples. Cytometry A. 2014;85(12):995–8. - PMC - PubMed
    1. Baumgart S, Peddinghaus A, Schulte‐Wrede U, Mei HE, Grätzkau A. OMIP‐034: comprehensive immune phenotyping of human peripheral leukocytes by mass cytometry for monitoring immunomodulatory therapies. Cytometry A. 2017;91(1):34–8. - PubMed

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