Journal: iScience

Article Title: Atypical B cells consist of subsets with distinct functional profiles

doi: 10.1016/j.isci.2023.108496

Figure Lengend Snippet:

Article Snippet: One day prior to sorting B cells, wells of a 96-well plate were each seeded with 30,000 adherent, CD40L-expressing 3T3 cells (kind gift from Dr. Mark Connors, NIH) in 100 μl IMDM/Glutamax/ 10% FBS containing 2× MycoZap Plus-PR (Lonza #VZA-2021), 100 ng/ml human IL-2 (GoldBio #1110-02-50), and 100 ng/ml human IL-21 (GoldBio #1110-21-10) to promote expansion and differentiation of B cells into antibody-secreting cells.

Techniques: Staining, Recombinant, Cell Isolation, Multiplex Assay, Sequencing, Plasmid Preparation, Software

Journal: The Journal of Experimental Medicine

Article Title: Activating receptors promote NK cell expansion for maintenance, IL-10 production, and CD8 T cell regulation during viral infection

doi: 10.1084/jem.20082387

Figure Lengend Snippet: IL-10 production by NK cells from MCMV-infected Prf1 −/− mice. (A) The top dot plots show the purity of NK cells (CD49b + ) and non–NK cells (CD49b − ) isolated from day 4 MCMV-infected wt and Prf1 −/− mice after CD49b + magnetic isolation. The numbers indicate percentages of cells in each box. Cytokine production of IFN-γ and IL-10 in the serum of infected wt and Prf1 −/− mice, or in 24 h conditioned media prepared with total splenic leukocytes and isolated subsets were determined. (B) The top dot plots show purity of NK cells (CD49b + Ly49H + ) and non–NK cells (CD49b − Ly49H − ) cells of day 4 MCMV-infected Prf1 −/− mice after flow sorting. The numbers indicate percentages of cells in each box. Cytokine production of IFN-γ and IL-10 in serum of infected Prf1 −/− mice, or in 24 h conditioned media from total splenic leukocytes and isolated subsets were determined. (C) Expression of IL-10 , IL-21 , and IL-15 in the total and sorted populations from day 4 MCMV-infected Prf1 −/− mice was analyzed by semiquantitative RT-PCR. Gapdh was used as an internal control. (D) Total splenic leukocytes, highly purified NK, and non–NK subsets from day 4 MCMV-infected Prf1 −/− mice were stimulated either on anti-Ly49H antibody or with cytokines. After 24 h of incubation, IL-10 production was determined in conditioned media. Results are representative of at least two independent experiments using a total of two to four individual mice. The studies in A were repeated twice for wt and four times for Prf1 −/− mice. Those in B were repeated three times, and those in C were repeated twice. The studies in D with anti-Ly49H antibody were repeated twice, and those with cytokines were repeated four times. The dashed white line indicates that intervening lanes have been spliced out. φ, not detected.

Article Snippet: For further stimulation, 10 5 highly purified cells were incubated either on control antibody (mouse IgG1)– and anti-Ly49H antibody–coated plates or with 100 ng/ml of mouse recombinant IL-21 (>97% purity; R&D Systems) in the presence or absence of 50 ng/ml IL-15 (>95% purity; R&D Systems).

Techniques: Infection, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Purification, Incubation