cd56 cells enrichment  (Miltenyi Biotec)

Journal: Frontiers in Immunology

Article Title: Identification and Isolation of Type II NKT Cell Subsets in Human Blood and Liver

doi: 10.3389/fimmu.2022.898473

Figure Lengend Snippet: Flow cytometry gating strategy for quantification of human type II NKT cells. (A) Human T2NKT cells were defined as CD3 + CD56 + CD161 + TCRγδ - TCRVα7.2 - TCRVα24 - lymphocytes. Gating of human PBMC resulted in a very low frequency of circulating T2NKT cells (0.3% of total CD3 + T cells). (B) Gating of human intrahepatic lymphocytes (IHL) revealed a higher frequency of T2NKT cells in non-steatotic liver (6.5% of total CD3 + T cells). Intrahepatic T2NKT cells could be subdivided into CD4 + and CD8 + cells. (C) Frequency of human T2NKT cells with respect to CD3 + T cells from 3 PBMC and 3 liver samples (steatosis 0%, n = 2; steatosis 15%, n = 1).

Article Snippet: Hence, we determined that enriching CD56 + cells using REAlease CD56 MicroBead Kit from Miltenyi prior to positive selection of CD3 + cells using CD3 MicroBeads from Miltenyi is an optimally efficient method for pre-enriching human T2NKT cells from blood ( ) .

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Identification and Isolation of Type II NKT Cell Subsets in Human Blood and Liver

doi: 10.3389/fimmu.2022.898473

Figure Lengend Snippet: Two-step magnetic-activated cell sorting (MACS) strategy for enrichment of human T2NKT cells. ( A ) Two-step MACS strategy consisted first in a CD56 positive selection from human PBMC with CD56 REAlease microbeads. These beads were then detached from the cell surface after separation. In the second step, NKT cells were enriched using CD3 microbeads. The outcome of this procedure was a CD3 + CD56 + cell isolate enriched in NKT cells. (B) Summary table indicating the frequency of cells relative to preMACS values and absolute (Abs) cell numbers after each MACS step. (C) Gating of human leucocytes enriched for CD56 + CD3 + cells using the two-step MACS protocol. The sample was enriched for iNKT cells (2% of total CD3 + T cells) and T2NKT cells (5.3% of total CD3 + T cells). (D) Expression of the transcription regulator promyelocytic leukemia zinc finger (PLZF) in subsets of human lymphocytes. Differential expression of PLZF was observed between conventional T cells (blue line) and NK cells (black line). There was similar PLZF expression in T2NKT cells (red line) and NK cells, whereas iNKT cells (green line) showed higher expression. (E) A CD4 + FoxP3 + CD25 + CD127 low subpopulation of CD3 + CD56 + CD161 + TCRγδ - TCRVα7.2 - TCRVα24 - T2NKT cells after the two-step enrichment process was identified in all 3 donors (one donor is shown as an example).

Article Snippet: Hence, we determined that enriching CD56 + cells using REAlease CD56 MicroBead Kit from Miltenyi prior to positive selection of CD3 + cells using CD3 MicroBeads from Miltenyi is an optimally efficient method for pre-enriching human T2NKT cells from blood ( ) .

Techniques: FACS, Selection, Expressing, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Identification and Isolation of Type II NKT Cell Subsets in Human Blood and Liver

doi: 10.3389/fimmu.2022.898473

Figure Lengend Snippet: In vitro expansion of FoxP3 + T2NKT cells. (A) Gating strategy to flow-sort T2NKT cell after double-step MACS enrichment. From left to right, cells were gated for lymphocytes based on FSC/SSC and singlets. Biotin FITC excluded cells labeled with TCRVα7.2, TCRVα24 or TCRγᵹ antibody. Cells were then gated for CD3 + CD4 + CD56 + CD161 + . (B) Schematic map depicting each step of the expansion protocol. (C) Cell counting after 3 weeks expansion using Flow Cytometry Counting Beads. Counting beads were positive for PE-Cy7, cells were separated from MACSiBeads based on FSC. (D) Quality control analysis of T2NKT cell expansion after 3 weeks. Lymphocytes were separated from MACSiBeads and dead cells by FCS/SSC. MAIT cells, γᵹ T cells and iNKT cells were excluded by staining with Biotin FITC. Within the CD3 + T cell population, PLZF was highly expressed (80.7% of the total lymphocyte population). These cells were mainly CD56 + CD161 + (75.7% of the total lymphocyte population) and a subset expressed regulatory-like phenotype FoxP3 + CD25 + CD127 low (31% of the total lymphocyte population). (E) Summary table indicating absolute number of cells before expansion (day 0) and after expansion (day 21). Percentage of FoxP3 + cells after expansion is included.

Article Snippet: Hence, we determined that enriching CD56 + cells using REAlease CD56 MicroBead Kit from Miltenyi prior to positive selection of CD3 + cells using CD3 MicroBeads from Miltenyi is an optimally efficient method for pre-enriching human T2NKT cells from blood ( ) .

Techniques: In Vitro, Labeling, Cell Counting, Flow Cytometry, Control, Staining

Journal: Frontiers in Immunology

Article Title: Adaptive Natural Killer Cells Integrate Interleukin-18 during Target-Cell Encounter

doi: 10.3389/fimmu.2017.01976

Figure Lengend Snippet: Interleukin (IL)-18 drives costimulation of adaptive natural killer (NK) cells. (A) IL12RB2 and (B) IL18RAP transcript abundance relative to GAPDH in ex vivo FACS-purified immature conventional NK cells (CD56 dim CD57 − NKG2C − ), mature conventional NK cells (CD56 dim CD57 + NKG2C − ), adaptive NKG2C + NK cells (CD56 dim CD57 + NKG2C + ), naïve T cells (CD3 + CD8 + CD45RA + CD45RO − CCR7 + ), and effector-memory T cells (CD3 + CD8 + CD45RA − CD45RO + CCR7 − ), n = 6 individual donors. Box plots display minimum to maximum and median. Statistical analysis performed with Friedman and Dunn’s multiple comparison test. (C) Representative staining of NF-κB(pS529) in FACS-purified adaptive NKG2C + NK cells after treatment with medium, phorbol-12-myristat-13-acetat (PMA), or interleukin (IL)-18 for 15 min. (D) Summary of frequencies of NF-κB(pS529) + cells either treated with medium or IL-18 over time, n = 5 donors. Symbols indicate mean and error bars SEM. Statistical analysis performed with repeated-measures two-way ANOVA with Bonferroni correction. (E,F) NK cells were cultured with K562/HLA-E in the presence of IL-12 + 18, IL-12, or IL-18. (E) Frequencies of ΔIFN-γ + [calculated by subtraction of the frequency of interferon (IFN)-γ + cells in the presence of K562/HLA-E alone] and (F) ΔTNF + adaptive NK cells, n = 12 donors. Symbols indicate mean and error bars SEM. Statistical analysis performed with Friedman and Dunn’s multiple comparison test. (G,H) NK cells were cultured with K562/HLA-E and varying concentrations of IL-18. (G) Frequencies of ΔIFN-γ + and (F) ΔTNF + adaptive NK cells, n = 6 donors. Symbols indicate mean and error bars SEM. Statistical analysis performed with Friedman and Dunn’s multiple comparison test. NS, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: To assess IFN-γ production upon stimulation with cytokines, CD56 + MACS-enriched cells were thawed, FACS-purified for viable CD3 − CD56 + NK cells, and cultured in complete medium in the presence or absence of 10 ng/mL human recombinant IL-12 (Miltenyi Biotec) and 100 ng/mL IL-18 (MBL) for 24 h. GolgiPlug and GoligStop (both BD Biosciences) were added according to the manufacturer’s instructions and present during the last 5 h of the stimulation.

Techniques: Ex Vivo, Purification, Comparison, Staining, Cell Culture