Journal: Frontiers in Immunology

Article Title: Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate

doi: 10.3389/fimmu.2022.803380

Figure Lengend Snippet: Ten-day expansion of NK cells from PBMCs with NK Cloudz and GMP human platelet lysate cultured in the G-Rex6M. (A) Flow cytometer density plots at 0, 7, and 10 days compare CD3 and CD56 expression of the expanded cell population. Black arrows indicate CD3 − CD56 + (NK) cells, which correspond with the green boxes in (B) the plot of cell distribution of viable CD45 + cells. (C) Viable NK cell fold change relative to day 0. (D) The viability of two populations: the light gray bars represent the viability of all CD45 + cells (including NK cells) and the dark gray bars represent the viability of only the CD45 + CD3 − CD56 + (NK) population. The graphs are broken out by donor (Dn A, Dn B, Dn C, and Dn D) to highlight the donor variability in the process.

Article Snippet: Samples were washed with flow buffer (PBS supplemented with 1% BSA (BP9700-100, ThermoFisher)), then stained with CD3-AlexaFluor 405 (FAB100V, Bio-Techne), CD56-APC (318310, Biolegend, San Diego, CA, USA), CD45-PE-cy7 (557748, BD Biosciences, Franklin Lakes, NJ, USA), CD16-PerCP (302030, Biolegend), NKp46-BB515 (564536, BD), NKG2D-PE (557940, BD), and CD2-AlexaFluor750 (FAB18561S, Bio-Techne).

Techniques: Cell Culture, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate

doi: 10.3389/fimmu.2022.803380

Figure Lengend Snippet: Comparison of the xeno-free 10% GMP human platelet lysate/NK Cloudz protocol to the existing FBS/NK Cloudz protocol in the G-Rex6M. The results show (A) the cell distribution on day 10, (B) NK (CD3 − CD56 + ) fold change between days 0 and 10, (C) the viability of either all CD45 + cells or NK cells alone on day 10, and (D) the percent of target K562 cells killed on day 10 with a 1:1 effector:target (E:T) ratio and a 4 h incubation. Data represent the mean ± SD from 4 separate donors. The GMP human platelet lysate resulted in similar purity, expansion, viability, and cytotoxicity as the FBS protocol.

Article Snippet: Samples were washed with flow buffer (PBS supplemented with 1% BSA (BP9700-100, ThermoFisher)), then stained with CD3-AlexaFluor 405 (FAB100V, Bio-Techne), CD56-APC (318310, Biolegend, San Diego, CA, USA), CD45-PE-cy7 (557748, BD Biosciences, Franklin Lakes, NJ, USA), CD16-PerCP (302030, Biolegend), NKp46-BB515 (564536, BD), NKG2D-PE (557940, BD), and CD2-AlexaFluor750 (FAB18561S, Bio-Techne).

Techniques: Comparison, Incubation

Journal: Frontiers in Immunology

Article Title: Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate

doi: 10.3389/fimmu.2022.803380

Figure Lengend Snippet: The G-Rex6M/NK Cloudz™ “low-touch” protocol compared with the flask-based “high-touch” feeder cell protocol. The feeder cell protocol used the same donors but was otherwise different: (See methods in (A) or in the Materials and Methods section). The results show the following: (B) the cell distribution on day 10, (C) the NK (CD3 − CD56 + ) fold change between days 0 and 10, (D) the viability of either all CD45 + cells (light gray bars) or NK cells alone (dark gray bars) on day 10, and (E) the percent of target K562 cells killed with a 1:1 effector:target (E:T) ratio over 4 h. Data represent the mean ± SD from 3 separate donors.

Article Snippet: Samples were washed with flow buffer (PBS supplemented with 1% BSA (BP9700-100, ThermoFisher)), then stained with CD3-AlexaFluor 405 (FAB100V, Bio-Techne), CD56-APC (318310, Biolegend, San Diego, CA, USA), CD45-PE-cy7 (557748, BD Biosciences, Franklin Lakes, NJ, USA), CD16-PerCP (302030, Biolegend), NKp46-BB515 (564536, BD), NKG2D-PE (557940, BD), and CD2-AlexaFluor750 (FAB18561S, Bio-Techne).

Techniques:

Journal: Heliyon

Article Title: The unilateral involution in the thymus of a 96-year-old male leads to the preservation of structural integrity in one thymic lobe, as assessed by the expression of medullar and cortical antigens and the presence of CD3+ cells

doi: 10.1016/j.heliyon.2022.e11734

Figure Lengend Snippet: The mixed population of CD3+/CD4-and CD3+/CD4+ T cells associate with the right lobe medullar thymic epithelium in the thymus of a 96-year-old male body donor. (a & b) show the immunostaining of the thymic sections with antibodies to CD3 (a) and CK-14 (b) antigens; an overlay of (a & b) is shown in (c). (d–i) show the immunostaining of the thymic sections with antibodies to CD3 (d & g) and CD4 (e & h) antigens. The overlay of (d & e) is shown in (f); the overlay of (g & h) is shown in (i). The white and green arrows in (f) show CD3+/CD4-and CD3+/CD4+ T cells, respectively; the arrows in (i) show the co-localization of CD3 and CD4 antigens in CD3+/CD4+ cells. The inset in (h) shows the nuclear stain with Hoechst 33342. Images in (a–f) and (g–i) were obtained under 10x and 40x magnification, consecutively, and represent a tissue section from the same block as shown in Figure 3. (j-l) show respectively the isotype controls for monoclonal antibodies used in all experiments discussed in the manuscript. (j) normal mouse IgG1 conjugated to Alexa Fluor 405 represents a control to immunostaining in Fig. 4 a, d and g. (k) normal mouse IgG1 conjugated to Alexa Fluor 488 represents a control to immunostaining in Fig. 3 a & d. (l) normal mouse IgG1 conjugated to Alexa Fluor 594 represents a control to immunostaining shown in Figures b & e and b, e & h. The scale bar: 50 μm.

Article Snippet: Directly labeled mouse monoclonal antibodies to cytokeratin 14 (AlexaFluor-594), cytokeratin 8 (AlexaFluor-488), CD3 (AlexaFluor-405), CD4 (AlexaFluor-546), FoxN1 (AlexaFluor-488), and isotype control normal mouse IgG1 conjugated to Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 405 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunostaining, Staining, Blocking Assay, Bioprocessing, Control

Journal: Cell reports

Article Title: Co-immunization of DNA and Protein in the Same Anatomical Sites Induces Superior Protective Immune Responses against SHIV Challenge

doi: 10.1016/j.celrep.2020.107624

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antigen-specific T cells were measured by intracellular cytokine staining followed by polychromatic flow cytometry using the following cocktail of cell surface antibodies: CD3-APC Cy7 (clone SP34–2, Cat#557757, BD PharMingen), CD4-V500 (clone L200, Cat#561488, BD), CD8-AlexaFluor-405 (clone 3B5, Cat#MHCD0826, Invitrogen), CD95FITC (clone DX2, Cat#556640, BD), CD28-PerCPCy5.5 (clone CD28.2, Cat#302922, Biolegend).

Techniques: Recombinant, Biomarker Assay, Software, Hybridization, Infection, Binding Assay, Spectrophotometry