easyseptm mouse cd8+ t cell enrichment kit  (STEMCELL Technologies Inc)

Journal: NPJ Precision Oncology

Article Title: Single-cell analysis reveals CD34 + CD90 + endothelial cells promote tumor metastasis in gallbladder cancer

doi: 10.1038/s41698-025-01040-2

Figure Lengend Snippet: A Flow cytometry was used to detect CD34 + CD90 + ECs in 2 GBC tissues (PE: CD34, FITC: CD90). B Representative multiple immune staining images of CD34 and CD90 in GBC tissues. C Immune staining of CD34 and CD90 on GBC tissue microarray. D Analysis of the correlation between CD34 + or CD34 + CD90 + cell contents and patient survival prognosis and clinical characteristics. OS, overall survival time.

Article Snippet: Subsequently, FITC-conjugated CD90 antibody was added to the sorted CD34 + endothelial cells, and an EasySepTM Release Human FITC Positive Selection Kit (STEMCELL Technologies) was used to isolate CD34 + CD90 + endothelial cells.

Techniques: Flow Cytometry, Staining, Microarray

Journal: iScience

Article Title: HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production

doi: 10.1016/j.isci.2025.112879

Figure Lengend Snippet: Cell-to-cell contact and soluble priming factors on MDM are modulated by HIV-1 infection as well as NK cells (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture at a ratio of 1:1, according to the seeding density of CD14 + monocytes. After 24 h, NK-cell ligand expression on MDM was assessed. (B–D) MDM infection frequencies (CD4 − p24+ cells) after HIV-1 infection with 89.6 and THRO.c with and without 24 h NK-cell co-culture are depicted. Differences in infection frequencies between MDM with and without NK cell co-culture for each strain were analyzed using the Wilcoxon signed-rank test (89.6: n = 8; THRO.c: n = 9). Representative histogram of HLA-E (C) and HLA-ABC (D) surface expression on unstimulated MDM (left). Median fluorescence intensity (MdFI) of HLA-E and HLA-ABC on unstimulated, HIV-1-infected and bystander MDM is compared between MDM cultured without (NK-) or with NK cells (NK+) (right). Differences in MdFI values between infected and bystander MDM and between NK- and NK + MDM in each strain were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (unstained (us): n = 19; unstim.: n = 21; 89.6 NK+: n = 14; 89.6 NK-: n = 9; THRO.c NK+: n = 11; THRO.c NK-: n = 10). (E–G) Scheme showing the cytokine analysis of supernatants from 24 h MDM-NK cell co-culture supernatants. Soluble IL-15 (F), IL-23 (G), and IL-18 (H, left) concentrations in MDM-NK cell co-culture supernatants from HIV-1 89.6, THRO.c and LPS/IFNγ conditions compared to unstimulated co-cultures. Differences in cytokine concentrations to unstimulated were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (89.6: n = 4; THRO.c: n = 9; LPS/IFNγ: n = 10). Only significant p -values are shown. (H, right) Relation between IL-18 concentrations in 89.6- and THRO.c-infected MDM-NK co-cultures and the corresponding infection frequency. Spearman correlation coefficient was used to analyze the correlation between infection frequency and cytokine concentration (89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels A and E (B, C right, D) and panel H (C left) . n.s., nonsignificant. See also .

Article Snippet: EasySepTM Human NK Cell Enrichment Kit , StemCell Technologies , Cat#19055.

Techniques: Infection, Co-Culture Assay, Expressing, Fluorescence, Cell Culture, Concentration Assay

Journal: iScience

Article Title: HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production

doi: 10.1016/j.isci.2025.112879

Figure Lengend Snippet: Priming by HIV-1-infected macrophages does not change NK-cell phenotype (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture. After 24 h, NK cell activation and receptor expression was assessed. (B) Flow cytometric detection of the two major NK cell subsets (CD56 br CD16 − and CD56 dim CD16 + ). (C) Concatenated contour plots showing the determination of activated NK cells as measured via CD69 upregulation (left) and frequency of CD69 + NK cells in bulk NK cells after 24 h co-culture with MDM in the different conditions (unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9) (right). Representative contour plots and expression (MdFI) of (D) intracellular perforin and (E) granzyme B, (F) 2B4, and (G) NKG2D in CD56 br and CD56 dim NK cells after co-culture with MDM in the different conditions ( Perforin : unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9; Granzyme B, 2B4 and NKG2D : unstim.: n = 10; LPS/IFNγ: n = 10; 89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. (C–G) Differences in percent positives or MdFI values of the different conditions compared to unstimulated in each NK cell subset were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) and only significant p values are depicted. Stainings were performed with antibody panels B and G (C, D, E) or panel B (F, G) . n.s., nonsignificant. ICS, intracellular staining. See also .

Article Snippet: EasySepTM Human NK Cell Enrichment Kit , StemCell Technologies , Cat#19055.

Techniques: Infection, Co-Culture Assay, Activation Assay, Expressing, Staining

Journal: iScience

Article Title: HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production

doi: 10.1016/j.isci.2025.112879

Figure Lengend Snippet: In vitro HIV-1 infection of macrophages primes NK-cell effector function toward cytokine release (A) Experimental setup of assessment of NK cell function after MDM co-culture. After co-culture with HIV-1-infected (89.6 and THRO.c (TH)), LPS/IFNγ-stimulated, and unstimulated MDM for 24 h, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation, measured by CD107a surface expression, and cytokine production, measured by intracellular CCL4, TNF, and IFNγ expression, were assessed. (B) Concatenated flow cytometry contour plots displaying CD107 and CCL4 expression of NK cells after MDM co-culture with and without targets (left). Representative contour plots for TNF and IFNγ plotted against CCL4 and determination of cytokine-positive NK cells (right). (C) Frequencies of CD107a+, CCL4+, TNF+, and IFNγ+ bulk (CD56 br+dim ) NK cells in response to 721.221 targets after co-culture with MDM in the different conditions. Differences in the functional response to targets in the different conditions to the unstimulated MDM priming condition were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg), only significant FDR-adjusted p values are shown ( p < 0.05) (89.6: n = 14; THRO.c: n = 12; LPS/IFNγ: n = 13). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels C and F (B, C) . See also .

Article Snippet: EasySepTM Human NK Cell Enrichment Kit , StemCell Technologies , Cat#19055.

Techniques: In Vitro, Infection, Cell Function Assay, Co-Culture Assay, Cell Culture, Expressing, Flow Cytometry, Functional Assay

Journal: iScience

Article Title: HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production

doi: 10.1016/j.isci.2025.112879

Figure Lengend Snippet: KIR+ NKG2A+ NK cells from PLWH respond to macrophage priming, whereas the CD56 neg subset remains dysfunctional (A) Set-up of NK cell and MDM co-culture from peripheral blood of PLWH. After 24 h co-culture with LPS/IFNγ-stimulated (stimulation for 24 h prior to co-culture) and unstimulated MDM, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation (measured by CD107a), and cytokine production, measured by intracellular CCL4, TNF, and IFNγ staining were assessed. (B) Flow cytometry staining of phenotypic MDM markers after 9 days of differentiation. (C) Expression of pSTAT1 (MdFI) in MDM with and without LPS/IFNγ stimulation. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (D) Representative dot plot of the three different subsets of NK cells (CD56 br , CD56 dim and CD56 neg ) (left) and respective frequencies among bulk NK cells from PLWH (right) ( n = 7). (E) Frequencies of CD107a-, CCL4-, TNF-, and IFNγ-expressing bulk (CD56 br+dim+neg ) NK cells with or without 721.221 targets after MDM co-culture. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (F) Gating strategy for NKG2A, NKG2C, CD57, and KIRs (any KIR Boolean gate: KIR2DL1/S5 OR KIR2DL2/L3 OR KIR3DL1/S1) in CD56 br , CD56 dim , and CD56 neg NK cells from PLWH. (G) Heatmap showing median frequencies of CD107a-, CCL4-, TNF-, and IFNγ-positive NK cells from PLWH of indicated subsets after MDM co-culture in response to targets. Values were clustered unsupervised according to the CD107a response, corresponding frequencies of the detected subsets in total NK cells are depicted to the right ( n = 7). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panel E (B, C), panel F (D, E, G), or panels C and F (F) . See also .

Article Snippet: EasySepTM Human NK Cell Enrichment Kit , StemCell Technologies , Cat#19055.

Techniques: Co-Culture Assay, Cell Culture, Staining, Flow Cytometry, Expressing