pecy7/pe conjugated anti-mouse cd150  (Thermo Fisher)

Journal: iScience

Article Title: Inhibition of SRC-mediated integrin signaling in bone marrow niche enhances hematopoietic stem cell function

doi: 10.1016/j.isci.2022.105171

Figure Lengend Snippet: Treatment with Src inhibitor Saracatinib alters cell cycle status of HSCs in vivo (A) Schematic representation of the experiments performed to examine the effect of Saracatinib on HSC cycling and differentiation. Three doses of vehicle alone or Saracatinib (25 mg/kg), through oral gavage, were given on alternate days followed by hematology analysis of PB cells and flow cytometry of BM MNCs. (B) Quantitative RT-PCR analysis performed to quantify Sdf-1α transcript levels lin − CD45 − BM cells from vehicle or Saracatinib treated mice. Actb was used as the internal control and relative gene expression was plotted. n= 5, N= 10; Mann-Whitney test: ∗∗∗p < 0.001. (C) Comparison of SDF-1α secretion in the BM plasma of vehicle or Saracatinib treated mice. The samples from the two groups of mice were used for ELISA-based detection and quantification. Relative levels of protein in the two groups of samples is shown. n= 6, N= 12; t test: ∗∗∗p < 0.001. (D-F) Flow cytometry-based cell cycle analysis of the LSK cells from the BM MNCs of vehicle and Saracatinib treated mice. The LSK cells were analyzed for 7-AAD and Ki67 staining to quantify the proportion of cells in G 0 (D), G 1 (E), and SG 2 /M (F) stages of cell cycle. n= 6, N= 6-8; Mann Whitney test: ∗p < 0.05. (G) Flow cytometry plots for the analysis of cell cycle stages of HSC cells from the BM MNCs of vehicle and Saracatinib treated mice. The Sca-1 + c-kit + cells gated on lin − CD48 − cells (top panel) were further gated for CD150 + cells identifying the adult BM HSCs (middle panel). The HSCs were analyzed for DAPI and Ki67 staining to analyze the proportion of cells in different cell cycle stages. (H-J) Comparison of the proportion of HSCs in G 0 (H), G 1 (I), SG 2 /M (J) stages of cell cycle in control and Saracatinib treated mouse BM. n = 4, N= 8; Mann-Whitney test: ∗p < 0.05, ∗∗∗∗p < 0.0001, ns not significant. (K) Flow cytometry-based analysis of BM cells to quantify the frequency of BM HSCs in vehicle and Saracatinib treated mice. The BM lin - c-Kit + cells gated for Sca-1 + cells (LSK cells) were further analyzed for CD48 and CD150 expression for detecting LT-HSCs (CD48 − CD150 + cells; upper left), ST-HSCs (CD48 − CD150 - cells; bottom left), MPP2 (CD48 + CD150 + cells; upper right), and MPP3/4 (CD48 + CD150 - cells; bottom right). (L-O) Frequency of LT-HSCs (L), ST-HSCs (M), MPP2 (N), and MPP3/4 (O) among LSK cells in the BM of the mice treated with or without Saracatinib. Flow cytometry-based experiments were performed on BM MNCs, and quantifications were performed based on gates shown in adjacent panels (K). n= 9 Mann-Whitney test: ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001. (P) Comparison of the circulating hematopoietic progenitors in peripheral blood following Saracatinib treatment. Methylcellulose-based colony assays were performed to detect CFU-Cs in 200μL peripheral blood from Saracatinib treated or untreated mice. n = 3, t test: ns not significant p > 0.05. (Q-T) Flowcytometry-based detection of circulating hematopoietic stem and progenitor cells. Frequency of LT-HSCs (Q), ST-HSCs (R), MPP2 (S), and MPP3/4 (T) in the peripheral blood derived MNCs from mice treated with or without Saracatinib. n = 8; Mann-Whitney test: ns not significant p > 0.05.

Article Snippet: For the characterization of HSPC sub-populations, the BM/PB derived MNCs, and the harvested progeny of cultured LSK cells were labelled with APC/FITC conjugated anti-mouse lineage antibody cocktail, PE/APC conjugated anti-mouse c-Kit, BB700/PECy7 conjugated anti-mouse Sca-1, FITC/APC-eFluor 780/eFluor 450 conjugated anti-mouse CD48 and PECy7/PE conjugated anti-mouse CD150 antibodies (ebiosciences).

Techniques: In Vivo, Flow Cytometry, Quantitative RT-PCR, Control, Gene Expression, MANN-WHITNEY, Comparison, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Cell Cycle Assay, Staining, Expressing, Derivative Assay

Journal: iScience

Article Title: Inhibition of SRC-mediated integrin signaling in bone marrow niche enhances hematopoietic stem cell function

doi: 10.1016/j.isci.2022.105171

Figure Lengend Snippet: Saracatinib treatment has no direct impact on HSCs ex vivo (A) Phase contrast images of the clusters of progeny from sorted LSK cells cultured in serum-free medium with SCF and TPO with or without Saracatinib (SRB; 50 and 100nM) after 5 days. (B) Expansion in the total number of cells after 5 days of culture. Sorted LSK cells were cultured with or without Saracatinib and harvested cells were counted using hemocytometer. n = 5, ns indicates not significant with p > 0.05, Student’s t test. (C) Comparison of LSK cell frequency in the cells harvested after 5 days of culture with or without Saracatinib. Harvested cells were used to perform flow cytometry-based phenotyping of cells and frequency of LSK cells was examined and compared between the samples. n = 5, ns indicates not significant with p > 0.05, Student’s t test. (D) Flowcytometry plots for analysis of the cell cycle stages of LSK cells from the harvested progeny of cultured hematopoietic progenitors. The lin − cells gated in the MNC population were analyzed for Sca-1 and c-kit expression to detect LSK cells. The LSK cells were further analyzed for CD48 and CD150 expression to detect LT-HSCs (CD48 − CD150 + cells; upper left), ST-HSCs (CD48 − CD150 - cells; bottom left), MPP2 (CD48 + CD150 + cells; upper right), and MPP3/4 (CD48 + CD150 - cells; bottom right). (E) The LSK cells identified in upper panel E, were further analyzed for DAPI and Ki67 staining to quantify the proportion of cells in G 0 , G 1 , and S-G 2 /M stages of cell cycle. (F) Comparison of the proportion of HSPCs (LSK cells) in various stages of cell cycle. The cells harvested after 5 days of culture with or without Saracatinib (50 and 100nM) were used for flow cytometry-based detection of various cell cycle stages based on Ki67 and DAPI staining. n = 6, ns indicates not significant with p > 0.05 using Student’s t test. (G) Frequency of LT-HSCs, ST-HSCs, MPP2s, and MPP3/4s in the cells harvested after 5 days of culture of LSK cells with or without Saracatinib. Flow cytometry-based experiments were performed on the harvested cells, and quantifications were performed based on gates shown in D lower panel. n = 5, ns indicates data not significant with p > 0.05, Student’s t test. (H) Flow cytometry analysis of the progeny of LSK cells cultured for 5 days with or without Saracatinib. To identify the myeloid progenitors MNCs gated on lin − cells were first analyzed for c-kit and Sca-1 expression. The lin - c-Kit + Sca-1 - cells were analyzed for the expression of CD16/32 and CD34 to identify MEPs, GMPs and CMPs. (I) The lymphoid progenitor cells (CLPs) in the progeny of LSK cells after 5 days of culture was identified as CD127 lo CD135 + cells within the lin - c-Kit lo Sca-1 + population. (J) Frequency of CLPs, GMPs, CMPs, and MEPs (D) in the cells harvested after 5 days of culture started with sorted BM derived LSK cells with or without Saracatinib (0, 50 and 100nM). Flow cytometry-based experiments were performed on the harvested cells, and quantifications were performed based on gates shown in panels H and I. n = 6-7 mice, ns indicates data not significant with p > 0.05, Student’s t test.

Article Snippet: For the characterization of HSPC sub-populations, the BM/PB derived MNCs, and the harvested progeny of cultured LSK cells were labelled with APC/FITC conjugated anti-mouse lineage antibody cocktail, PE/APC conjugated anti-mouse c-Kit, BB700/PECy7 conjugated anti-mouse Sca-1, FITC/APC-eFluor 780/eFluor 450 conjugated anti-mouse CD48 and PECy7/PE conjugated anti-mouse CD150 antibodies (ebiosciences).

Techniques: Ex Vivo, Cell Culture, Comparison, Flow Cytometry, Expressing, Staining, Derivative Assay

Journal: iScience

Article Title: Inhibition of SRC-mediated integrin signaling in bone marrow niche enhances hematopoietic stem cell function

doi: 10.1016/j.isci.2022.105171

Figure Lengend Snippet: Faster recovery of hematopoietic system in mice following Saracatinib treatment (A) Schematic representation of radiation recovery experiments performed on vehicle and Saracatinib treated mice. Following sub-lethal irradiation, PB counts were counted weekly to compare the radiation recovery in the two groups of mice. After eight weeks, experiments were terminated and flow cytometry analysis was performed on BM cells. (B-J) Number of WBCs (B), lymphocytes (C), granulocytes (D), monocytes (E), eosinophils (F), platelets (G), RBCs (H), hematocrit values (HCT; I), and hemoglobin levels (HGB; J) were compared between control and Saracatinib injected groups, for a period of up to eight weeks. (K-O) Comparison of recovery of BM hematopoietic system from radiation injury in control versus Saracatinib treated mice. After 8 weeks of irradiation, the mice were sacrificed and BM mononuclear cells were analyzed for HSPC sub-populations. Frequency of LSK cells (K) and the four sub-populations, based on the expression of SLAM markers CD150 and CD48, were examined; CD150 − CD48 + LSK (MPP3/4; L), CD150 + CD48 + LSK (MPP2; M), CD150 − CD48 − LSK (ST-HSCs; N) and CD150 − CD48 + LSK (LT-HSCs; O) cells were identified and quantified. Data obtained from 10-12 independent biological replicates, was plotted as mean ± SEM ∗p < 0.05, ∗∗p < 0.01 by Mann-Whitney test.

Article Snippet: For the characterization of HSPC sub-populations, the BM/PB derived MNCs, and the harvested progeny of cultured LSK cells were labelled with APC/FITC conjugated anti-mouse lineage antibody cocktail, PE/APC conjugated anti-mouse c-Kit, BB700/PECy7 conjugated anti-mouse Sca-1, FITC/APC-eFluor 780/eFluor 450 conjugated anti-mouse CD48 and PECy7/PE conjugated anti-mouse CD150 antibodies (ebiosciences).

Techniques: Irradiation, Flow Cytometry, Control, Injection, Comparison, Expressing, MANN-WHITNEY

Journal: iScience

Article Title: Inhibition of SRC-mediated integrin signaling in bone marrow niche enhances hematopoietic stem cell function

doi: 10.1016/j.isci.2022.105171

Figure Lengend Snippet:

Article Snippet: For the characterization of HSPC sub-populations, the BM/PB derived MNCs, and the harvested progeny of cultured LSK cells were labelled with APC/FITC conjugated anti-mouse lineage antibody cocktail, PE/APC conjugated anti-mouse c-Kit, BB700/PECy7 conjugated anti-mouse Sca-1, FITC/APC-eFluor 780/eFluor 450 conjugated anti-mouse CD48 and PECy7/PE conjugated anti-mouse CD150 antibodies (ebiosciences).

Techniques: Purification, Virus, Recombinant, Western Blot, Membrane, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Isolation, cDNA Synthesis, SYBR Green Assay, CRISPR, Knock-Out, Gene Expression, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a ) Flowcytometry analysis of the BM cells derived from 16-week-old FVB/NJ (WT; upper panel) and Postn −/− (KO; lower panel) mice ( N =12). ( b ) Frequency of SLAM KLS cells per million BM cells derived from 16-week-old FVB/NJ (WT) and Postn −/− (KO) mice ( N =12, t test: * P <0.008). ( c , d ) BrdU incorporation assays to examine the proliferation status of KLS cells (ST-HSCs; c ) and SLAM KLS cells (LT-HSCs; d ) in WT and Postn −/− mice. BrdU staining in addition to HSC markers in BM cells following 3 ( c ) or 7 ( d ) days of BrdU infusion ( n =3, N =9, t test: * P <0.02). ( e ) Schematic representation of the competitive repopulation assays. 50,000 total BM cells derived from WT/ Postn −/− mice (CD45.2) were transplanted into sub-lethally irradiated Rag2 −/− γC −/− mice. PB chimerism was followed for 12 weeks, after which secondary transplantations were performed. ( f , g ) Donor-derived PB chimerism in primary ( f ) and secondary ( g ) recipients transplanted with total BM cells from 8-week-old WT/ Postn −/− mice ( n =3, N =18, t test: * P <0.03). ( h – m ) Blood obtained from 16-week-old wild-type (WT) and Postn −/− mice was assessed for WBC count ( h ), RBC count ( i ), haematocrit value ( j ), haemoglobin level ( k ), lymphocyte ( l ) and granulocytes ( m ) numbers ( N =12, t test: *** P <0.001, ** P <0.01, * P <0.05). ( n , o ) Donor-derived PB chimerism in primary ( n ) and secondary ( o ) recipients transplanted with sorted primitive HSCs (CD150 + CD48 − KLS cells) total BM cells from 16-week-old WT/ Postn −/− mice ( n =3, N =18, t test: * P =0.02). ( p ) Frequency of primitive HSCs in the donor-derived fraction of BM cells from secondary recipients ( n =3, N =6, t test: * P =0.007). ( q ) Proportion of donor-derived primitive HSCs in secondary recipients in G0 stage of cell cycle ( n =3, N =6, t test: ** P =0.001). ( n =independent experiments, N =number of mice. Error bars indicate mean±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Derivative Assay, BrdU Incorporation Assay, BrdU Staining, Irradiation

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a – c ) Following sub-lethal irradiation PB counts were measured weekly for 7 weeks. Numbers of WBCs ( a ), granulocytes ( b ), and haematocrit values ( c ) were compared between FVB/NJ (WT) and Postn −/− (KO) mice ( n =3, N =18, t test: ** P <0.01, * P <0.05). ( d ) Following sub-lethal irradiation the cell cycle status of BM derived KLS cells was analysed. Two weeks after irradiation, BM cells were isolated and flowcytometry analysis was performed to assess proliferation of lin − c-kit + Sca-1 + (KLS) cells with Hoechst labelling ( n =3, N =9). ( e , f ) Flowcytometry based analysis performed during 8 weeks following sub-lethal irradiation to compare the number of SLAM KLS ( e ) and various lineage committed ( f ) cells in the BM of FVB/NJ (WT) and Postn −/− (KO) mice ( n =3, N =9, t test * P <0.05). ( n =independent experiments, N =number of mice. Error bars indicate mean ±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Irradiation, Derivative Assay, Isolation

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a – c ) BM derived KLS cells were cultured for 5 days in serum-free medium with SCF and TPO in the absence (ST) or presence (STP) of Postn. ( a ) Cultured BM KLS cells harvested after 5 days were labelled with the PKH-26 dye and plated on ST2 cell feeders. The relative proportion of cells that adhered after 3 h was plotted ( n =4, t test: NS P >0.05). ( b ) Migration of cultured BM KLS cell progeny following 5 days of culture, and labelled with the PKH-26 dye, towards SDF-1α was assessed using a trans-well system. The relative proportion of cells that migrated after 3 h was plotted ( n =4, t test: NS P >0.05). ( c ) Cultured BM KLS cells harvested after 5 days were tested for their in vivo homing capacity, by infusing into lethally irradiated animals. The percentage of transplanted CFCs that homed into the BM within 16 h was plotted ( n =4, N =12, t test: NS P >0.05). ( d ) Expression of Itgav (CD51) and Itgab3 (CD61) on SLAM KLS cells assessed by flowcytometry ( n =6). ( e ) KLS cells incubated with SCF+TPO without (ST) or with (STP) Postn for 3 h were allowed to adhere on cyclo-RGDfK coated plates. The percentage of cells that adhered to the plates after 3 h was plotted for each condition ( n =4, t test: * P =0.018). ( f , g ) BM derived KLS cells were cultured in serum-free medium containing SCF and TPO for 2 days without (ST) or with (STP) Postn alone, or in combination with neutralizing antibodies against Itgav (STP+αItgav) or Itgb3 (STP+αItgb3) for 2–5 days. ( n =4, t test: ** P <0.01; scale bar, 50 μm). ( f ) Bright field image showing cell expansion after 2 days. The proliferating cells appear as a cluster of cells in the middle of the round-bottom 96-well plates. ( g ) After 5 days of culture, cells were harvested and fold expansion was compared. ( n =independent experiments, N =number of mice. Error bars indicate mean ±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Derivative Assay, Cell Culture, Migration, In Vivo, Irradiation, Expressing, Incubation

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a – d ) Counts for various blood cell types in Vav-iCre + ; Itgav fl/fl (KO), Vav-iCre + ; Itgav fl/+ (HT) and Vav-iCre + ; Itgav +/+ (WT) mice. WBC ( a ), monocyte ( b ), granulocyte ( c ) lymphocyte ( d ) counts in WT, HT and KO mice ( N =10, t test: ** P <0.01, * P <0.05). ( e – g ) Phenotypic analysis of BM cells by flowcytometry ( e ), to compare the numbers of KLS ( f ) and SLAM KLS ( g ) cells ( N =12, t test: ** P <0.01, * P <0.05). ( h ) Schematic representation of the competitive repopulation assay. 200 KLS cells or 10,000 WBMCs from Vav;Itgav −/− mice (CD45.2) along with 100,000 or 90,000 whole BM competitor cells (CD45.1), respectively, were transplanted into sub-lethally irradiated CD45.1 WT CD45.1 mice. PB chimerism was followed for 12 weeks, after which secondary transplantations were performed. ( i , j ) PB chimerism in primary and secondary recipients transplanted with 10,000 total BM cells from Vav-iCre + ; Itgav fl/fl (KO) and Vav-iCre + ; Itgav +/+ (WT) mice, together with 90,000 competitor cells ( n =3, N =15, t test: ** P <0.01, * P <0.05). ( k , l ) PB chimerism in primary and secondary recipients transplanted with 200 KLS cells from the BM of Vav-iCre + ; Itgav fl/fl (KO) and Vav-iCre + ; Itgav +/+ (WT) mice, together with 100,000 competitor cells ( n =3, N =12, t test: ** P <0.01, * P <0.05). ( n =independent experiments, N =number of mice. Error bars indicate mean ±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Irradiation

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a ). Representative primitive HSCs (SLAM KLS cells) isolated by FACS and stained with anti-γH2AX antibodies (pseudo-color red) and Hoechst 33342 (pseudo-color green). White arrows indicate foci. ( n =4). ( b ). Representative example of primitive HSCs (SLAM KLS cells) isolated by FACS and stained with anti-RPA antibodies (pseudo-color red) and Hoechst 33342 (pseudo-color green). White arrows indicate foci. ( n =4). ( c ). Percentage of HSCs with γH2AX-marks from young Postn −/− mice (right), young WT (left), and old WT (middle) mice. ( n =4, t test: * P <0.05). ( d ). Average number of γH2AX-positive foci in primitive HSCs from young Postn −/− mice (right), young WT (left) and old WT (middle) mice. ( n =4, t test: * P <0.05). ( n =independent experiments, Error bars indicate mean ±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Isolation, Staining