MBTP 35: Immunophenotyping of tumor-infiltrating lymphocytes from human ovarian carcinoma using flow cytometry

Miltenyi Biotec-tested panel 35 (MBTP 35)

This application protocol describes the characterization of human monocytes, neutrophils, eosinophils, and T, B, and NK lymphocyte populations as well as CD4⁺, CD8⁺, and CD56⁺CD3⁺ T cell subsets in human ovarian carcinoma using flow cytometry. Prior to staining the tumor was dissociated employing the gentleMACS™ Octo Dissociator with Heaters and Tumor Dissociation Kit, human.

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Protocol

Staining protocol

Surface staining

Volumes given below are for up to 10⁷ nucleated cells. When working with fewer than 10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁷ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

Cell number was determined.
Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
Cells were resuspended in 100 μL of buffer.
10 μL of the 8-Color Immunophenotyping Cocktail, human were added.
1 μL of 7-AAD Staining Solution was added.
Cells were resuspended and incubated for 10 minutes in the dark at 4 °C.
Cells were washed by addition of 1–2 mL of PEB buffer and centrifuged at 300×g for 10 minutes at 4 °C. Supernatant was aspirated completely.
Cells were resuspended in 100 µL of buffer before flow cytometric analysis using the MACSQuant® Analyzer.

Flow cytometric analysis

Gating strategy showing the analysis of the immune cell composition in a dissociated human ovarian carcinoma sample. A gate around viable cells (A) as well as a gate around single cells in forward scatter area (FSC-A) versus forward scatter height (FSC-H) (B) was set to eliminate doublets. To identify the major circulating blood cell types CD45 was used to target all leukocytes (C). These cells were further separated from debris via FSC and side scatter (SSC) (D). Monocytes were discriminated based on their CD14 expression (E). Among the non-monocyte population, B cells were defined as CD19⁺ (F). The remaining cells were separated into CD16⁺ SSC^high neutrophils, CD16^– SSC^high eosinophils as well as a CD16^–/dim SSC^low population (G). CD3 and CD56 were used to distinguish CD56⁺ NK cells, CD3⁺ T cells and a CD3⁺CD56⁺ T cell population (H). CD3⁺ T cells were divided into CD4⁺ helper T cells and CD8⁺ cytotoxic T cells (I).

Materials

Reagent and instrument requirements

Antibody panel details

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
CD3	REA613	T cells	PE	579/34 (blue)
CD4	REA623	T helper cells	Vio® Bright 667	667/30 (red)
CD8	REA734	Cytotoxic T cells	APC-Vio 770	785/62 (red)
CD14	REA599	Monocytes	VioBlue®	450/50 (violet)
CD16	REA423	Neutrophils, eosinophils	Vio Bright 515	525/50 (blue)
CD19	REA675	B cells	PE-Vio 770	785/62 (blue)
CD45	REA747	Leukocytes	VioGreen™	525/50 (violet)
CD56	REA196	NK cells	Vio Bright 515	525/50 (blue)
7-AAD Staining Solution	-	Viability	-	667/30 (blue)

Additional reagents used

PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.
8-Color Immunophenotyping Kit, human (# 130-120-640)
Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
(Optional) MACS MiniSampler Plus (# 130-105-745)
(Optional) Chill 5 Rack (# 130-092-951)
(Optional) MACSQuant Calibration Beads (# 130-093-607)
(Optional) MACS Comp Bead Kit, anti‑REA (# 130‑104‑693), anti-mouse Igκ (# 130‑097‑900), or anti-human Igκ (# 130‑104‑187)

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MBTP 35: Immunophenotyping of tumor-infiltrating lymphocytes from human ovarian carcinoma using flow cytometry

Miltenyi Biotec-tested panel 35 (MBTP 35)

Protocol

Materials

Protocol

Staining protocol

Surface staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

CD45 Antibody, anti-human, VioGreen™, REAfinity™

CD8 Antibody, anti-human, APC-Vio
^®
770
, REAfinity™

CD14 Antibody, anti-human, VioBlue
^®
, REAfinity™

CD3 Antibody, anti-human, PE, REAfinity™

CD19 Antibody, anti-human, PE-Vio
^®
770
, REAfinity™

CD4 Antibody, anti-human, Vio
^®
Bright R667
, REAfinity™

CD56 Antibody, anti-human, Vio
^®
Bright B515
, REAfinity™

CD16 Antibody, anti-human, Vio
^®
Bright B515
, REAfinity™

8-Color Immunophenotyping Kit, human

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