Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: A Correlation analyses between NK cell proportion and age ( n = 26). B Correlation analyses between NK cell numbers and age ( n = 26). C Correlation analyses between purity of the NK cell product reinfused into each individual and age ( n = 26). The spearman correlation analysis was used in all cases. The correlation was significant at 0.05 (two-sided). D CD3 + T cells were separated and SA-β-gal staining was performed pre- and post-infusion of NK cells ( n = 26). E Expression of senescence (P16 Ink4a and P21) (n = 25) and SASP markers (IL-6, monocyte chemoattractant protein-1 [MCP-1], plasminogen activator 1 [Pai1]) ( n = 26) in CD3 + T cells were analysed by qRT-PCR. Differences were assessed by the two-tailed paired Student’s t -test. ns P > 0.05, * P < 0.05, ** P < 0.01. Two parallel samples were set in each experiment and three independent experiments were performed for each result.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Staining, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: Flow cytometry to detect expression of ( A ) perforin and ( B ) CD69 was performed after 48-h co-incubation with NK cells and human adipose tissue, treated with conditional medium from control (CON) or senescent (SEN) preadipocytes for 24 h. Cells were gated in CD3 - CD56 + location. n = 3. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. * P < 0.05, ** P < 0.01. C Senescent markers for P16 and P21 were detected by Western blotting. n = 3. D Secreted proinflammatory cytokines in conditional medium from adipose tissue was collected 48-h after removing the NK cells. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ns P > 0.05, * P < 0.05, ** P < 0.01. Two parallel samples were set in each experiment and three independent experiments were performed for each result.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Flow Cytometry, Expressing, Incubation, Control, Two Tailed Test, Western Blot

Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: Flow cytometry to detect the expression of ( A ) CD69 and ( B ) IFN-γ was performed after a 24 h co-incubation with mouse NK cells and preadipocytes or irradiation-induced preadipocytes. n = 3. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. ** P < 0.01. C SNC apoptosis was detected after a 24-h co-incubation with DiR-labeled NK cells by flow cytometry. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ** P < 0.01. D Activating ligands and inhibitory ligands of NK cells on preadipocytes, doxorubicin-induced preadipocytes or irradiation-induced preadipocytes were measured by qPCR. n = 3. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. * P < 0.05 (Irradiation vs CON). # P < 0.05 (Doxorubicin vs CON). E 1 × 10 6 DiR-labeled control preadipocytes or irradiation-induced senescent preadipocytes were implanted into the abdominal cavity. Representative images showing fluorescence activity in mice seven days after 5 × 10 6 NK cell treatment. Quantification of fluorescence activity. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ** P < 0.01, **** P < 0.0001. F Quantification of cytotoxicity of non-senescent and senescent counterparts induced by irradiation or adriamycin incubated with increasing NK cells for 24 h. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Two parallel samples were set in each experiment and three independent experiments were performed for each result.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Flow Cytometry, Expressing, Incubation, Irradiation, Two Tailed Test, Labeling, Control, Fluorescence, Activity Assay

Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: A , B Cytotoxicity of mouse NK cells against SNCs supplemented with different concentrations of DA after a 24-h co-incubation. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. C Data showing cAMP content in NK cells after 24-h incubation with SNCs supplemented with different concentrations of DA. n = 3. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. * P < 0.05, *** P < 0.001, **** P < 0.0001. D Western blot analysis of phosphorylated CREB level in NK cells after 24-h incubation with SNCs supplemented with different concentrations of DA. E DR expression in NK cells was detected after co-incubation with SNCs or control cells by qPCR. n = 3. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. * P < 0.05, ** P < 0.01. F , G Cytotoxicity of mouse NK cells against SNCs supplemented with 10 −9 M DA and SCH23300 or haloperidol after a 24-h co-incubation. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ns P > 0.05, * P < 0.05, ** P < 0.01. H cAMP content in NK cells after incubation with SNCs supplemented with 10 −9 M DA and SCH23300 or haloperidol after a 24-h co-incubation. n = 3. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. ns P > 0.05, **** P < 0.0001. I Western blot analysis of phosphorylated CREB level in NK cells after incubation with SNCs supplemented with 10 −9 M DA and SCH23300 or haloperidol after a 24-h co-incubation. Two parallel samples were set in each experiment and three independent experiments were performed for each result.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Incubation, Western Blot, Expressing, Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: A Peripheral dopamine levels in young (10-week-old) and aged (20-month-old) mouse. n = 6. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. * P < 0.05. B Peripheral dopamine levels induced by 10 mg/kg Acein 1 h after Acein administration in old mice. n = 5. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. ** P < 0.01. C , D NK cells in peripheral blood were detected by flow cytometry after a four-month treatment. n = 4. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. ns P > 0.05, * P < 0.05, ** P < 0.01. E CD69 expression level in NK cells. n = 4. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. ns P>0.05, * P < 0.05, *** P < 0.001. F SA- β -gal staining of adipose tissue in the groin. G Expression levels of senescent markers P16 and P21 in adipose tissue in the groin. H BrdU was injected intraperitoneally at 24 and 72 h before euthanasia. BrdU + Ki67 + adipocytes in the groin were detected by flow cytometry. n = 4 . Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. ns P > 0.05, * P < 0.05. Two parallel samples were set in each experiment.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Two Tailed Test, Flow Cytometry, Expressing, Staining, Injection

Journal: Cell Proliferation

Article Title: Serum‐free media for the production of human mesenchymal stromal cells: a review

doi: 10.1111/cpr.12063

Figure Lengend Snippet: Various commercially available serum‐free media

Article Snippet: Table summarizes various commercially available appropriate SFM. table ft1 table-wrap mode="anchored" t5 Table 4 caption a7 S. no. Serum‐free media/product name Cat no. Company Media type References cited 1 BD MosaicTM hMSC serum‐free medium (kit) 355701 BD Biosciences CD, SF 25 CellGro TM 24803–0500 CellGenix SF, PE 38 3 HEScGRO SCM020 Merck Millipore CD, SF, ACF 88 Mesenchymal stem cell growth medium DXF C‐28019 PromoCell CD, SF, XF Not cited MesenCult ® ‐XF (culture kit) 5429 Stem Cell Technologies CD, SF, ACF 82 , 89 , 90 , 91 , 92 , 93 , 94 , 95 6 MesenGro ZRD‐MGro‐500 StemRD CDM 38 7 MSC Qualified PLus TM PLS2 Compass Biomedical XF Not cited 8 MSC‐Gro TM (SF, complete) SC00B3 Vitro Biopharma SF, CG 38 9 MSCGS‐ACF (MSC supplements) 7572 ScienCell Research SF 38 10 mTeSR 5850 Stem Cell Technologies SF 32 11 PRIME‐XV TM MSC Expansion SFM 31000 Irvine Scientific SF Not cited 12 RS‐Novo TM and GEM‐Novo N/App Kerry Bio‐Sciences CD, SF, ACF Not cited 13 SPE‐IV SPE‐IV‐EBM/500 Abecell‐Bio SF 38 14 Stemline MSC expansion medium S1569 Sigma Alderich CD, SF, AF 96 , 97 15 StemPro ® MSC SFM (media kit) A10332‐01 Life Technologies CD, SF, 30 , 81 , 98 , 99 16 StemPro ® MSC SFM‐XF (media kit) A10675‐01 Life Technologies CD, SF, AF 31 , 82 , 100 , 101 , 102 17 StemXVivo TM CCM014 R&D Systems, Inc. SFM 38 18 STK2 N/App Two Cells Co., Ltd. CD, SF, AF 103 , 104 19 TheraPEAK TM MSCGM‐CD TM (Bullet kit) 190632 Lonza CD, SF, AF 33 , 82 , 103 , 104 20 Ultrasor G (lyophilized) 15950‐017 Pall Biosepra SF 105 Open in a separate window AF, animal component‐free; Cat no., catalogue number; CD, chemically defined; CG, clinical grade (especially for MSC clinical trials); PE, preclinical ex vivo use only; PF, protein‐free; SF, serum‐free; XF, xeno‐free.

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