Journal: Molecular Vision

Article Title: Lithium chloride promotes host resistance against Pseudomonas aeruginosa keratitis

doi:

Figure Lengend Snippet: Apoptosis in the infected corneas assessed with flow cytometry. A : The apoptotic ratios in lithium chloride– (LiCl-) versus sodium chloride (NaCl)–treated corneas at 1 and 5 days p.i. were detected with propidium iodide (PI) staining associated with flow cytometry, as calculated by the average percentage of PI-positive cells in the infected cornea ( D ). The respective apoptotic ratios of macrophages ( B ) and neutrophils ( C ) in LiCl- versus NaCl-treated corneas at 1 and 5 days p.i. were analyzed with flow cytometry associated with triple staining for PI, F4/80, and Gr-1, as calculated by the average percentage of PI-positive cells in either F4/80-positive macrophages ( E ) or Gr-1-positive neutrophils ( F ). G : The percentages of the macrophages and neutrophils in the corneal infiltrating cells were determined with F4/80 and Gr-1 staining associated with flow cytometry. Data are the mean±standard error of the mean (SEM) and represent three individual experiments (n=5). ***, p<0.001.

Article Snippet: After blocking, cells were distributed into 100 μl samples and incubated with the following Abs for 30 min on ice: allophycocyanin (APC)-conjugated anti-Gr-1 (BD, Sparks, MD), Alexa Fluor 488-conjugated anti-F4/80 (Invitrogen), or isotype control APC-conjugated antirat IgG2b (BD) and Alexa Fluor 488-conjugated antirat IgG2a (Invitrogen).

Techniques: Infection, Flow Cytometry, Staining

Journal: STAR Protocols

Article Title: Protocol for isolation and analysis of the leukemia stem cells in BCR-ABL-driven chronic myelogenous leukemia mice

doi: 10.1016/j.xpro.2023.102123

Figure Lengend Snippet: Antibody panel for flow cytometry analysis of myeloid leukemia cells

Article Snippet: APC anti-mouse Gr-1 antibody , BD Biosciences , Cat#553129; RRID: AB_398532.

Techniques: Flow Cytometry, Marker

Journal: STAR Protocols

Article Title: Protocol for isolation and analysis of the leukemia stem cells in BCR-ABL-driven chronic myelogenous leukemia mice

doi: 10.1016/j.xpro.2023.102123

Figure Lengend Snippet:

Article Snippet: APC anti-mouse Gr-1 antibody , BD Biosciences , Cat#553129; RRID: AB_398532.

Techniques: Recombinant, Cell Culture, Flow Cytometry

Journal: Nature Communications

Article Title: A PI3K p110β–Rac signalling loop mediates Pten-loss-induced perturbation of haematopoiesis and leukaemogenesis

doi: 10.1038/ncomms9501

Figure Lengend Snippet: ( a ) Survival of Pten Δ/Δ (blue; n =9; median survival=26 days), Pten Δ/Δ ;p110α Δ/Δ (green; n =9; median survival=31 days), Pten Δ/Δ ;p110β Δ/Δ (red; n =9; median survival=55 days), Pten Δ/Δ ;p110δ −/− (orange; n =7; median survival=37 days) and control animals (black) represented as DPI. The log-rank test was used to compare survival between groups. ( b ) Survival time and the development of MPN and T-ALL in each group of mice as indicated. Survival of Pten Δ/Δ ( n =9; median survival=26 days), Pten Δ/Δ ;p110α Δ/Δ ( n =9; median survival=31 days), Pten Δ/Δ ;p110β Δ/Δ ( n =9; median survival=55 days), Pten Δ/Δ ;p110δ −/− ( n =7; median survival=37 days) and control animals (black) represented as DPI. Black dots represent MPN disease cases and red dots represent T-ALL cases in each group. The log-rank test was used to compare survival between groups. ( c ) Histopathology of the spleen (× 1 and × 40) and liver (× 100) taken at 26 DPI. Increased splenomegaly and disruption of spleen architecture as well as increased myeloid cell infiltration after Pten deletion. Scale bar, 5 μm. ( d ) Total cellularity of the spleen is represented as mean±s.d. of n =5 except Pten Δ/Δ ;p110δ −/− ( n =10). Two-way ANOVA test was applied to compare the spleen cellularity. ( e ) Representative flow cytometry plots of Mac1 + Gr1 + cells in the spleen. Samples were analysed at 26 DPI. Bar graphs represent the mean±s.d. of n =9 ( Pten Δ/Δ ;p110δ −/− ), 10 ( Pten Δ/Δ ;p110α Δ/Δ ), 11 (ctrl) or 13 ( Pten Δ/Δ and Pten Δ/Δ ;p110β Δ/Δ ). Two-way ANOVA test was applied to compare the Mac1-Gr1 cells in the spleen. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Antibodies used for flow cytometry were directly coupled and directed against B220 (APC, BD Pharmingen), cKit (PE-Cy7, BioLegend), CD3 (PE-Cy7, BD Bioscience), CD4 (APC-H7, BD Pharmingen), CD8 (ECD, Beckman Coulter), CD11b (PE, BD Bioscience), CD16/32 (PE, eBioscience), CD34 (FITC, BD Pharmingen), CD45.1 (FITC, BD Pharmingen), CD45.2 (PerCP-Cy5.5, BD Pharmingen), CD48 (APC-Cy7, BD Pharmingen), CD127 (ECD, BD Pharmingen), CD150 (PerCP-Cy5.5, BioLegend), Gr1 (APC-Alexa 700, BD Bioscience), lineage cocktail (APC, BD Pharmingen), Sca1 (Brilliant Violet 421, BioLegend), Ki67 (Alexa 488, BD Pharmingen).

Techniques: Histopathology, Flow Cytometry

Journal:

Article Title: E2F1 and E2F2 Determine Thresholds for Antigen-Induced T-Cell Proliferation and Suppress Tumorigenesis

doi: 10.1128/MCB.21.24.8547-8564.2001

Figure Lengend Snippet: Loss of E2F1 and E2F2 results in hyperproliferation of hematopoietic progenitors. (A) Bone marrow cells from 6-week-old male littermates of the indicated genotypes were harvested and stained with either fluorescently labeled anti-B220, anti-GR-1, anti-CD34, or anti-TER119, fixed, and then stained with PI. Cell cycle profiles (determined by PI intensity) of the different bone marrow subsets gated for the expression of the indicated marker are shown. The percentage of cells in either the G1 (first solid peak), S (hatched), or G2 (second solid peak) phase was determined using the ModFit program. The percentage of cells in S phase is indicated. The y axis represents cell number, and the x axis represents PI fluorescence intensity. This experiment is representative of more than four experiments. (B) Bone marrow cells from mice of the indicated genotypes were harvested and cultured with 1μM BrdU in 10% FBS–IMDM medium for 2 h. The cells were collected and stained with fluorescently labeled anti-BrdU. BrdU incorporation was detected by flow cytometry. In three similar experiments, DKO bone marrow cells exhibited a 2.5 ± 0.5 (standard error [SE])-fold increase in BrdU incorporation compared to bone marrow from E2F1+/− E2F2+/− littermates. (C) Bone marrow cells were harvested from Rag2−/− mice of the indicated E2F1/E2F2 genotypes (the right three panels are from littermates) and analyzed as in A for cell cycle profiles in the B220+ subset.

Article Snippet: The following Pharmingen antibodies were used: phycoerythrin (PE)-linked α-CD4, Cy-Chrome-α-CD8, PE-α-CD25, biotin-α-CD25 (together with streptavidin-Cy-Chrome), allophycocyanin (APC)-linked α-B220, APC-α-Thy1.2, APC-α-GR-1, APC-α-Ter119, APC-α-CD34, PE-α-CD43, fluorescein isothiocyanate (FITC)-linked TCR variable-chain beta (Vβ) 5, FITC-TCR Vβ6, and FITC-CD44.

Techniques: Staining, Labeling, Expressing, Marker, Fluorescence, Cell Culture, BrdU Incorporation Assay, Flow Cytometry