cd14 monocyte isolation  (Miltenyi Biotec)

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: Phenotypic changes of monocyte-derived macrophages after in vitro HIV-1 infection (A) Differentiation of monocyte-derived macrophages (MDM) from CD14 + monocytes of HIV-1(−) donors using M-CSF for seven days and HIV-1 infection with strains 89.6 and THRO.c for six days or stimulation with LPS and IFNγ for 48 h; stainings were performed at day 11 of MDM differentiation. (B) Representative flow cytometry staining of phenotypic MDM markers and gating for HIV-1-infected MDM (CD4 − p24+). (C) Representative immunofluorescent images of unstimulated and HIV-1-89.6-infected MDM stained for Iba-1 (yellow), HIV-1 p24 (magenta), and DAPI (cell nuclei, cyan). Scale bar: 50 μm. For staining antibody information see Table S2. (D) MDM infection frequencies 6 days after HIV-1 infection with 89.6 ( n = 9) and THRO.c ( n = 11). (E) Representative histograms of surface CD80 expression on MDM and log 2 fold change to unstimulated. (LPS/IFNγ: n = 13; 89.6: n = 9; THRO.c: n = 11). (F) Histograms of intracellular pSTAT1 staining and log 2 fold change to unstimulated. (LPS/IFNγ: n = 3; 89.6: n = 7). (G) Contour plot of pSTAT1 in MDM infected with 89.6 (left). RFI values of HIV-1-infected (CD4 − p24+) and bystander (CD4 + p24-) MDM (middle). Relation between pSTAT1 relative fluorescence intensity (RFI) in HIV-1-infected MDM (CD4 − p24+) and the corresponding infection frequency (right). Spearman correlation coefficient was used to analyze the correlation between infection frequency and RFI of pSTAT1 ( n = 7). (H and I) Representative histograms and log 2 fold change to unstimulated of CD48 (LPS/IFNγ: n = 13; 89.6: n = 9; THRO.c: n = 11) and NKG2D ligands (MIC-A/B, ULBP2/5/6) compared to unstimulated MDM (LPS/IFNγ: n = 10; 89.6: n = 5; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. (E, F, H, I) Differences were analyzed using the Wilcoxon signed-rank test against the value 0 with FDR correction (Original Benjamini and Hochberg). Donor numbers vary due to stringent technical exclusion criteria (see Methods). Stainings were performed with antibody panels A and E (B, D, E, H), panel A (I NKG2DL-BV650 ) or panel E (F, G, I NKG2DL-APC) . See also and .

Article Snippet: Peripheral blood mononuclear cells (PBMC) from HIV-1(-) donors were isolated from peripheral blood anticoagulated with EDTA using density gradient centrifugation, washed with Hank’s Balanced Salt Solution (HBSS), and directly subjected to positive CD14 + monocyte isolation (StemCell Technologies, #17858) following manufacturer's instructions with the exception of reducing the amount of selection cocktail used to 50% of the recommended amount.

Techniques: Derivative Assay, In Vitro, Infection, Flow Cytometry, Staining, Expressing, Fluorescence

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: Peripheral blood mononuclear cells (PBMC) from HIV-1(-) donors were isolated from peripheral blood anticoagulated with EDTA using density gradient centrifugation, washed with Hank’s Balanced Salt Solution (HBSS), and directly subjected to positive CD14 + monocyte isolation (StemCell Technologies, #17858) following manufacturer's instructions with the exception of reducing the amount of selection cocktail used to 50% of the recommended amount.

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