ram34  (Miltenyi Biotec)

Journal: STAR Protocols

Article Title: Protocol for the isolation and characterization of mouse alveolar bone marrow hematopoietic stem cells

doi: 10.1016/j.xpro.2025.103875

Figure Lengend Snippet: Flow cytometric analysis and gating strategy for identifying hematopoietic stem cells Representative flow cytometry plots showing the sequential gating strategy for identifying HSCs in the alveolar bone marrow. (Gate 1) FSC-A vs. SSC-A to identify cells. (Gate 2) Singlet-FSC discrimination. (Gate 3) Singlet-SSC discrimination. (Gate 4) Live cell gating. (Gate 5) CD45+ hematopoietic cells. (Gate 6) Lineage-negative cells. (Gate 7) KSL (c-Kit+Sca-1+Lin−) cells. (Gate 8) CD34−CD150+KSL HSCs. The numbers indicate the percentage of cells in each gate. The plots shown are representative of data obtained from male mice.

Article Snippet: FITC anti-mouse CD34 (clone RAM34) (1:100) , eBioscience , Cat# 11-0341-85; RRID: AB_465022.

Techniques: Flow Cytometry

Journal: Endocrinology

Article Title: Weight Gain With Advancing Age Is Controlled by the Muscarinic Acetylcholine Receptor M4 in Male Mice

doi: 10.1210/endocr/bqaf064

Figure Lengend Snippet: List of primary and secondary antibodies used for immunofluorescence studies

Article Snippet: CD34 , Anti-CD34 Monoclonal Antibody (RAM34), FITC , Thermo Fisher Scientific , 11-0341-81 , AB_465021 , 1/100.

Techniques: Immunofluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: IL-33/ST2 signaling in ILC2s drives exhaustion and myeloid skewing of HSCs in response to hematopoietic stress and aging

doi: 10.1016/j.isci.2025.112378

Figure Lengend Snippet: IL-33/ST2 signaling in ILC2s promotes myelopoiesis (A) BM ILC2 numbers in Rora flox/flox ( n = 12) and Rora flox/flox x IL7RCre (ΔILC2, n = 11) mice (pooled data from 3 independent experiments). (B) Total BM cell numbers in Rora flox/flox and ΔILC2 mice. (C–E) Total BM hematopoietic stem and progenitor cell (HSPC, C), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), megakaryocyte-erythroid progenitor (MEP, D), and myeloid cell (CD11b+, E) numbers in Rora flox/flox and ΔILC2 mice. (F) Representative histogram of the ST2 expression on ILC2s, measured by flow cytometry; black: ST2 −/− mouse; orange: BL/6. (G) IL-33 BM fluid levels in BL/6 ( n = 11) and IL-33 −/− ( n = 3) mice, and IL-33 serum levels in BL/6 mice ( n = 5) measured with a U-Plex multiplex assay from MSD (pooled data from 3 independent experiments). (H) Results of the BL/6-IL-33 −/− chimera experiment. 10 weeks post-transplantation, recipients were sacrificed, and the BM fluid was analyzed for IL-33. (I) Representative histogram of the IL-33 expression on PDGFR-α + sca-1 + mesenchymal stromal cells (PαS cells), measured in BL/6 (black line) and IL-33-GFP-reporter mice (IL-33 rep ; red line) by flow cytometry. (J) ILC2 numbers in BL/6 ( n = 17), ST2 −/− ( n = 14), and IL-33-knockout (IL-33 −/− , n = 14) mice (pooled data from 6 independent experiments). (K–N) Total BM cell (K), HSPC (L), CMP, GMP, MEP (M), and myeloid cell (N) numbers in BL/6 ( n = 13–17), ST2 −/− ( n = 14) and IL-33 −/− ( n = 9–14) mice. (O and P) Colony forming assay performed with fluorescence-activated cell sorting (FACS)-purified HSPCs from naive BL/6 or ST2 −/− mice, in the presence or absence of recombinant murine (rm)IL-33 (100 ng/ml). The first plating was performed with 1 × 10 3 primary HSPCs. The colonies were counted (O), and the number of cells per colony was evaluated (P) 7 days post-plating. 1 × 10 4 cells were replated in methylcellulose with or without rmIL-33 (100 ng/ml). The replating was analyzed 7 days post-plating ( n = 3 per condition). (Q–T) BL/6 vs. ST2 −/− competitive chimera experiment. (Q) Experimental setup for the competitive chimera experiment: 1.5 × 10 4 Ly5.1 + 5.2 + BL/6 and 1.5 × 10 4 Ly5.2+ ST2 −/− HSPCs were co-transplanted into lethally irradiated (2 × 60cGy) Ly5.1 + BL/6 ( n = 4, males) or ST2 −/− ( n = 5, males) recipients. The chimerism was assessed 18 weeks post-transplantation. (R) Total BM cell numbers of BL/6 and the ST2 −/− recipients 18 weeks post-transplantation. (S and T) Percent distribution (chimerism) of ILC2 (S), total BM cells, HSPC, and myeloid cells (T) 18 weeks post-transplantation. Data are represented as mean ± SD. Statistics: two-tailed Student’s t test (A, B, and E), one-way ANOVA (I–L); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: FITC anti-mouse CD34 (RAM34) , eBioscience , Cat#11-0341-82; RRID: AB_465021.

Techniques: Expressing, Flow Cytometry, Multiplex Assay, Transplantation Assay, Knock-Out, Fluorescence, FACS, Purification, Recombinant, Irradiation, Two Tailed Test

Journal: iScience

Article Title: IL-33/ST2 signaling in ILC2s drives exhaustion and myeloid skewing of HSCs in response to hematopoietic stress and aging

doi: 10.1016/j.isci.2025.112378

Figure Lengend Snippet: Transfer of ST2-competent ILC2s into ST2 −/− recipients normalizes myelopoiesis (A) Experimental layout of the LMPP transfer experiment. Lymphoid-primed multipotent progenitor cells (lin − sca-1 + cKit + CD135 + LMPPs) were FACS-sorted from Rag1 −/− x CD45.1 donors and then transplanted into sub-lethally (450 cGy) irradiated BL/6 and ST2 −/− recipients. 18 weeks post-transplantation, the BM compartment was analyzed. (B) FACS-gating strategy for BM-resident CD45.1 + ILC2s. (C) Ly5.1 + ILC2 numbers in the BM of Ly5.2 + BL/6 ( n = 11) and ST2 −/− ( n = 11) mice. (D) Total BM cell numbers in BL/6 ( n = 11) and ST2 −/− ( n = 13) LMPP-recipients, and BL/6 ( n = 12) and ST2 −/− ( n = 13) control mice 18 weeks post-transplantation (pooled data from 4 independent experiments). (E–G) BM CD45.2 + HSPC (E), CMP, GMP (F), and myeloid cell numbers (G) in BL/6 and ST2 −/− LMPP-recipients and control mice 18 weeks post-transplantation. (H–K) Rora flox/flox and ΔILC2 mice were used as recipients of Rag1 −/− x CD45.1 LMPPs in a similar experimental setup (pooled data from 2 independent experiments). (H) Total BM cell numbers in Rora flox/flox ( n = 7) and ΔILC2 ( n = 8) LMPP-recipients and Rora flox/flox ( n = 6) and ΔILC2 ( n = 7) control mice 18 weeks post-transplantation. (I-K) BM CD45.2 + HSPC (I), CMP, GMP (J), and myeloid cell (K) numbers in Rora flox/flox and ΔILC2 LMPP-recipients and control mice 18 weeks post-transplantation. Data are represented as mean ± SD. Statistics: two-tailed Student’s t test; ∗ p < 0.05, ∗∗ p < 0.01. See also Figure S2 .

Article Snippet: FITC anti-mouse CD34 (RAM34) , eBioscience , Cat#11-0341-82; RRID: AB_465021.

Techniques: Irradiation, Transplantation Assay, Control, Two Tailed Test

Journal: iScience

Article Title: IL-33/ST2 signaling in ILC2s drives exhaustion and myeloid skewing of HSCs in response to hematopoietic stress and aging

doi: 10.1016/j.isci.2025.112378

Figure Lengend Snippet: IL-33/ST2 signaling in ILC2s promotes myelopoiesis (A) BM ILC2 numbers in Rora flox/flox ( n = 12) and Rora flox/flox x IL7RCre (ΔILC2, n = 11) mice (pooled data from 3 independent experiments). (B) Total BM cell numbers in Rora flox/flox and ΔILC2 mice. (C–E) Total BM hematopoietic stem and progenitor cell (HSPC, C), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), megakaryocyte-erythroid progenitor (MEP, D), and myeloid cell (CD11b+, E) numbers in Rora flox/flox and ΔILC2 mice. (F) Representative histogram of the ST2 expression on ILC2s, measured by flow cytometry; black: ST2 −/− mouse; orange: BL/6. (G) IL-33 BM fluid levels in BL/6 ( n = 11) and IL-33 −/− ( n = 3) mice, and IL-33 serum levels in BL/6 mice ( n = 5) measured with a U-Plex multiplex assay from MSD (pooled data from 3 independent experiments). (H) Results of the BL/6-IL-33 −/− chimera experiment. 10 weeks post-transplantation, recipients were sacrificed, and the BM fluid was analyzed for IL-33. (I) Representative histogram of the IL-33 expression on PDGFR-α + sca-1 + mesenchymal stromal cells (PαS cells), measured in BL/6 (black line) and IL-33-GFP-reporter mice (IL-33 rep ; red line) by flow cytometry. (J) ILC2 numbers in BL/6 ( n = 17), ST2 −/− ( n = 14), and IL-33-knockout (IL-33 −/− , n = 14) mice (pooled data from 6 independent experiments). (K–N) Total BM cell (K), HSPC (L), CMP, GMP, MEP (M), and myeloid cell (N) numbers in BL/6 ( n = 13–17), ST2 −/− ( n = 14) and IL-33 −/− ( n = 9–14) mice. (O and P) Colony forming assay performed with fluorescence-activated cell sorting (FACS)-purified HSPCs from naive BL/6 or ST2 −/− mice, in the presence or absence of recombinant murine (rm)IL-33 (100 ng/ml). The first plating was performed with 1 × 10 3 primary HSPCs. The colonies were counted (O), and the number of cells per colony was evaluated (P) 7 days post-plating. 1 × 10 4 cells were replated in methylcellulose with or without rmIL-33 (100 ng/ml). The replating was analyzed 7 days post-plating ( n = 3 per condition). (Q–T) BL/6 vs. ST2 −/− competitive chimera experiment. (Q) Experimental setup for the competitive chimera experiment: 1.5 × 10 4 Ly5.1 + 5.2 + BL/6 and 1.5 × 10 4 Ly5.2+ ST2 −/− HSPCs were co-transplanted into lethally irradiated (2 × 60cGy) Ly5.1 + BL/6 ( n = 4, males) or ST2 −/− ( n = 5, males) recipients. The chimerism was assessed 18 weeks post-transplantation. (R) Total BM cell numbers of BL/6 and the ST2 −/− recipients 18 weeks post-transplantation. (S and T) Percent distribution (chimerism) of ILC2 (S), total BM cells, HSPC, and myeloid cells (T) 18 weeks post-transplantation. Data are represented as mean ± SD. Statistics: two-tailed Student’s t test (A, B, and E), one-way ANOVA (I–L); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: eFluor450 anti-mouse CD34 (RAM34) , eBioscience , Cat#48-0341-82; RRID: AB_2043837.

Techniques: Expressing, Flow Cytometry, Multiplex Assay, Transplantation Assay, Knock-Out, Fluorescence, FACS, Purification, Recombinant, Irradiation, Two Tailed Test

Journal: iScience

Article Title: IL-33/ST2 signaling in ILC2s drives exhaustion and myeloid skewing of HSCs in response to hematopoietic stress and aging

doi: 10.1016/j.isci.2025.112378

Figure Lengend Snippet: Transfer of ST2-competent ILC2s into ST2 −/− recipients normalizes myelopoiesis (A) Experimental layout of the LMPP transfer experiment. Lymphoid-primed multipotent progenitor cells (lin − sca-1 + cKit + CD135 + LMPPs) were FACS-sorted from Rag1 −/− x CD45.1 donors and then transplanted into sub-lethally (450 cGy) irradiated BL/6 and ST2 −/− recipients. 18 weeks post-transplantation, the BM compartment was analyzed. (B) FACS-gating strategy for BM-resident CD45.1 + ILC2s. (C) Ly5.1 + ILC2 numbers in the BM of Ly5.2 + BL/6 ( n = 11) and ST2 −/− ( n = 11) mice. (D) Total BM cell numbers in BL/6 ( n = 11) and ST2 −/− ( n = 13) LMPP-recipients, and BL/6 ( n = 12) and ST2 −/− ( n = 13) control mice 18 weeks post-transplantation (pooled data from 4 independent experiments). (E–G) BM CD45.2 + HSPC (E), CMP, GMP (F), and myeloid cell numbers (G) in BL/6 and ST2 −/− LMPP-recipients and control mice 18 weeks post-transplantation. (H–K) Rora flox/flox and ΔILC2 mice were used as recipients of Rag1 −/− x CD45.1 LMPPs in a similar experimental setup (pooled data from 2 independent experiments). (H) Total BM cell numbers in Rora flox/flox ( n = 7) and ΔILC2 ( n = 8) LMPP-recipients and Rora flox/flox ( n = 6) and ΔILC2 ( n = 7) control mice 18 weeks post-transplantation. (I-K) BM CD45.2 + HSPC (I), CMP, GMP (J), and myeloid cell (K) numbers in Rora flox/flox and ΔILC2 LMPP-recipients and control mice 18 weeks post-transplantation. Data are represented as mean ± SD. Statistics: two-tailed Student’s t test; ∗ p < 0.05, ∗∗ p < 0.01. See also Figure S2 .

Article Snippet: eFluor450 anti-mouse CD34 (RAM34) , eBioscience , Cat#48-0341-82; RRID: AB_2043837.

Techniques: Irradiation, Transplantation Assay, Control, Two Tailed Test