human and mouse purified cells  (AllCells LLC)

Journal: European journal of immunology

Article Title: Tracking in vivo dynamics of NK cells transferred in patients undergoing stem cell transplantation.

doi: 10.1002/eji.201444586

Figure Lengend Snippet: Figure 3. Analysis of counts, proliferation, and HLA of recipient NK cells. (A) Mean (solid line) ± SEM (dotted line) of frequency of Ki67+ NK cells (left y-axis, gray) and absolute NK-cell counts/μL (right y-axis, black) in three to seven recipients (r) at different time points after SCT. NK cells were gated as viable (LD−), CD45+, CD3−, CD14−, CD19−lineage (Lin)−CD56+ lymphocytes as shown in Supporting Infor- mation Figure 3. Asterisks represent mean ± SEM values of five healthy related family donors (d). Values were determined by FC after gat- ing on viable CD45+Lin−CD56+ NK cells in PBMCs (for Ki67) or whole blood (for absolute NK-cell number). Expression values were linearly interpolated between measurement time points. Data are pooled from seven to three patients, depending on survival, each assessed at max- imal nine different time points. (B) Expression of Ki67 among NK cells from one representative patient of seven total patients (gated as in A). (C and D) NK cells (gated as in A) were analyzed by FC of PBMCs isolated from one representative recipient out of three at indicated time points after haploSCT. Expression of HLA-A02, displayed by donor (d) but not by recipient (r) NK cells was determined by FC. Gated HLA-A02+ and HLA-A02−NK cells were analyzed for expression of indicated markers.

Article Snippet: Stimulation of NK cells MACS-purified CD56+ cells were isolated using CD56 microbeads (Miltenyi Biotec) and cultured for 16 h at 37°C and 5% CO2 in RPMI (Gibco) containing 10% human AB serum (Lonza), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (PAA Laboratories) in the presence of 10 μg/mL brefeldin A (Sigma) FITC anti-CD107a (H4A3; BD Biosciences) and 0.7 μL/mL GolgiStop (BD Biosciences).

Techniques: Expressing, Isolation

Journal: European journal of immunology

Article Title: Tracking in vivo dynamics of NK cells transferred in patients undergoing stem cell transplantation.

doi: 10.1002/eji.201444586

Figure Lengend Snippet: Figure 4. Phenotypic and functional analysis of recipient NK cells. (A) Geometrical MFI (geoMFI) of CD56 and frequency of the indicated markers among viable (LD−) CD45+Lin−CD56+ NK cells, gated as shown in Supporting Information Figure 3, during the course of reconstitution are plotted as mean (black solid line) ± SEM (black dotted lines) of three to seven recipients (r), depending on survival, each assessed at maximal nine different time points, after receiving CD34+ SCT and NK-cell transfer. Expression values were determined by FC and linearly interpolated between measurement time points. Mean frequency of Ki67+ NK cells (as in Fig. 3A) is plotted as gray solid line in all graphs. Mean ± SEM of geoMFI (CD56) and frequency of each indicated marker of five healthy related donors (d) is plotted as black asterisk. Self-specific KIR2DL1 and KIR3DL1 were defined according to the presence of HLA-C2 or HLA-Bw4 in the respective donors. (B and C) CD107a expression induced by actR stimulation was measured by FC in MACS-enriched CD56+ NK cells, gated as described in (A), at the indicated time points. Plotted are (B) mean + SEM of CD107a+

Article Snippet: Stimulation of NK cells MACS-purified CD56+ cells were isolated using CD56 microbeads (Miltenyi Biotec) and cultured for 16 h at 37°C and 5% CO2 in RPMI (Gibco) containing 10% human AB serum (Lonza), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (PAA Laboratories) in the presence of 10 μg/mL brefeldin A (Sigma) FITC anti-CD107a (H4A3; BD Biosciences) and 0.7 μL/mL GolgiStop (BD Biosciences).

Techniques: Functional Assay, Expressing, Marker

Journal: European journal of immunology

Article Title: Tracking in vivo dynamics of NK cells transferred in patients undergoing stem cell transplantation.

doi: 10.1002/eji.201444586

Figure Lengend Snippet: Figure 5. PCA of NK cells in healthy donors and one patient undergoing haploSCT with- out NK-cell transfer. (A) Percentage of viable (LD−) CD45+Lin−CD56+ NK cells, gated as shown in Supporting Information Figure 3, expressing the indicated markers was assessed by FC at eight different time points in one patient after haploSCT without NK-cell trans- fer. (B and C) PCA was calculated using viable CD45+Lin−CD56+ NK cells from five healthy donors, using expression values (transformed fluorescence) for CD16, NKG2A, CD62L, and CD56. (B) A representative heat map of marker contributions to the PCs calculated in healthy donors is shown (n = 5) and (C) representative scatter plots of PC scores color coded for the indicated markers, with increasing expression values from blue (minimal) to red (maximal) is presented. (D) Scatter plot of healthy donor PC scores as depicted in (C), with red line joining mean PC1 and PC2 scores of CD45+Lin−CD56+

Article Snippet: Stimulation of NK cells MACS-purified CD56+ cells were isolated using CD56 microbeads (Miltenyi Biotec) and cultured for 16 h at 37°C and 5% CO2 in RPMI (Gibco) containing 10% human AB serum (Lonza), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (PAA Laboratories) in the presence of 10 μg/mL brefeldin A (Sigma) FITC anti-CD107a (H4A3; BD Biosciences) and 0.7 μL/mL GolgiStop (BD Biosciences).

Techniques: Expressing, Transformation Assay, Marker

Journal: European journal of immunology

Article Title: Tracking in vivo dynamics of NK cells transferred in patients undergoing stem cell transplantation.

doi: 10.1002/eji.201444586

Figure Lengend Snippet: Figure 6. PCA and FC analysis of NK cells from patients receiving CD34+ SCT with or without NK-cell transfer. (A) Mean PC1 scores at different time points after SCT in one patient receiving CD34+ SCT without NK-cell transfer (red) and one representative patient with NK-cell transfer (blue) out of three. (B–D) Viable (LD−) CD45+Lin−CD56+ NK cells from one patient receiving CD34+ SCT without NK-cell transfer (red) or from one patient receiving CD34+ SCT in combination with NK-cell transfer (blue) were analyzed during the (B) first, (C) second, or (D) third week after SCT. Expression of the indicated markers was analyzed by FC and percentage of marker expression on NK cells, gated as shown in Supporting Information Figure 3, in the patients with and without NK-cell transfer is summarized in (B) and (C).

Article Snippet: Stimulation of NK cells MACS-purified CD56+ cells were isolated using CD56 microbeads (Miltenyi Biotec) and cultured for 16 h at 37°C and 5% CO2 in RPMI (Gibco) containing 10% human AB serum (Lonza), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (PAA Laboratories) in the presence of 10 μg/mL brefeldin A (Sigma) FITC anti-CD107a (H4A3; BD Biosciences) and 0.7 μL/mL GolgiStop (BD Biosciences).

Techniques: Expressing, Marker