Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Intracellular and intranuclear staining can be improved with extended incubation times. (A) Representative staining obtained in 30 min versus 16 hr. (B) Stain indices for Foxp3 and IL‐2. Mouse splenocytes were stimulated and stained for intracellular cytokines as described in the Methods section. Data were acquired on a Cytek Aurora Spectral Cytometer.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Staining, Incubation, Cytometry

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Increasing incubation time reduces batch effects. (A) Representative staining and MFI of SIGLEC‐8 on CD45 + SSC hi CD16 ‐ cells from the same donor over 3 independent experiments. (B) Representative staining and MFI of CD123 on CD45 + SSC lo CD3 − CD19 − CD14 − CD16 − cells from the same donor over 3 independent experiments. (C) Human whole blood immunophenotyping data from the same donor over three independent experiments, stained for 30 min or 16 hr. tSNE plots were generated using the parameters CD45, SSC‐A, CD4, CD8, CD127, CD16, CD19, CD3, CD123, CD20, CD25, Fcer1a, CD11c, SIGLEC‐8, CD56, CD14, and HLA‐DR. Data were acquired on a BD LSRFortessa cytometer. FlowSOM clusters are shown in a colored overlay. (D) Cross entropy distances between samples stained for 30 min or 16 hr. (E) Mouse data from four experiments over the course of two years. Data were acquired on a BD FACSymphony A5 cytometer. tSNE plots were generated using the parameters CD4, CD8, Foxp3, CD103, Neuropilin, CD44, CD62L, Ki67, ICOS, PD‐1, CTLA‐4, CD25, KLRG1, CD69, ST2, and Helios on CD3 + T cells. FlowSOM clusters are shown in a colored overlay. (F) Cross entropy distances between mouse samples (intra‐batch variation) or batches (inter‐batch variation). Significance was tested by unpaired t‐test.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Incubation, Staining, Generated, Cytometry

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Titration is essential to maximize sensitivity. (A) Titration of CD3‐Spark Blue 550. (B) Intracellular overnight staining for PD‐1 on viable CD4 + CD3 + T cells at the indicated dilutions on cells fixed and permeabilized with the eBioscience Foxp3 Fix/Perm kit.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Titration, Staining

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Controls confirm specificity is maintained with overnight staining. (A) IL‐2 staining on WT or IL‐2‐deficient mouse CD4 + T cells. (B) pSTAT5 and Foxp3 staining on mouse CD4 + T cells with or without IL‐2 stimulation.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Staining

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Reagents Used for This Study

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Concentration Assay

Journal: Bioengineering

Article Title: RNU ( Foxn1 RNU -Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats

doi: 10.3390/bioengineering8010012

Figure Lengend Snippet: CD68+ staining is increased in RNU DMM-treated sites compared to Sprague Dawley. CD68 staining was quantified and showed increased levels in RNU rats ( A ). Yet, CD163 ( B ) and FoxP3 ( C ) were unaltered. CD4 staining was increased in RNU rats ( D ) while CD8 staining was lower ( E ). In addition, CD4, CD8, and FoxP3 levels were low. DRAQ5 showed no change in DNA staining ( F ). Data shown are means ± SEM of 8 animals. Letters not shared indicated a significant difference ( p < 0.05, Tukey).

Article Snippet: Primary antibodies used in this experiment were: mouse anti-Pax7 (ab55494, Abcam, Cambridge, UK); rabbit anti-nicotinic acetylcholine receptor-epsilon (AChR-ε, ab65180, Abcam); mouse anti-nicotinic acetylcholine receptor-gamma (AChR-γ, MA3-043, Thermo Fisher Scientific, Waltham, MA, USA); mouse anti-myosin heavy chain-fetal (fMyHC, SC-53097, Santa Cruz Biotechnology); CD68 (ab125212, Abcam); CD163 (ab87099, Abcam); CD8 (MAB116, R&D Systems), CD4 (MAB554, R&D Systems), FoxP3 (MAB8214, R&D Systems) and were diluted in PBS with 1% BSA and 0.3% Tween-20.

Techniques: Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Serine protease inhibitor from the muscle larval Trichinella spiralis ameliorates non-alcoholic fatty liver disease in mice via anti-inflammatory properties and gut-liver crosstalk.

doi: 10.1016/j.biopha.2024.116223

Figure Lengend Snippet: Fig. 4. rTs-SPI attenuated macrophage infiltration and promoted CD4þCD25þFoxp3þ Tregs population in NAFLD mice. (A) Macrophage infiltration and (B) CD4+CD25+Foxp3+ Tregs in spleen using Flow cytometry analysis; Quantitative analysis of (C) F4/80+ macrophages and (D) CD4+CD25+Foxp3+ Tregs in spleen; (E) Microphotographs show total macrophages staining with F4/80 (red) and DAPI (blue) in liver (600x); (F) Quantitative analysis of F4/80+ macrophages in liver; The hepatic mRNA expression levels of (G) F4/80 and (H) Foxp3. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For Treg analysis, cells were stained with Percp/cy5.5 anti-mouse CD4 and APC anti-mouse CD25 antibodies (Elabscience, China), followed by fixing and permeabilizing using a Foxp3/transcription factor staining kit (Elabscience, China) and staining with PE anti-mouse Foxp3 antibody (Elabscience, China).

Techniques: Flow Cytometry, Staining, Expressing

Journal: The Indonesian Biomedical Journal

Article Title: 2-(3-(chloromethyl)benzoyloxy)benzoic Acid Increases CD4+ Regulatory T-Cell Population and FoxP3 Expression in Lipopolysaccharide-induced Mice

doi: 10.18585/inabj.v15i4.2429

Figure Lengend Snippet: Figure 3. Foxp3-expression in splenocytes of LPS- induced mice. Mice were induced with/without LPS and treated orally with/without ASA or 3-CH2Cl. Spleen of the mice were isolated, processed, and the splenic FoxP3 expression was measured with flow cytometry as described in Methods. A: Representative histogram with MFI value obtained from each group. B: MFI value of each group. **0.01≥p>0.001; ***0.001≥p>0.0001, tested with upaired t-test.

Article Snippet: Ten μg of APC Anti-Mouse Foxp3 Antibody (Elabscience) was added to the splenocytes.

Techniques: Expressing, Isolation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Safe and Efficient Sigma1 Ligand: A Potential Drug Candidate for Multiple Sclerosis

doi: 10.3390/ijms231911893

Figure Lengend Snippet: List of monoclonal antibodies used for flow cytometric analysis.

Article Snippet: FoxP3 Antibody, anti-human, APC , Miltenyi Biotec , 130-125-580.

Techniques: Bioprocessing

Journal: Nature Communications

Article Title: Tracing immune cells around biomaterials with spatial anchors during large-scale wound regeneration

doi: 10.1038/s41467-023-41608-9

Figure Lengend Snippet: a The unsupervised clustering indicated the anatomical structure of samples. b Gene enrichment analysis between ECM_LW and Ctrl_LW group. c Violin plot showing the up-regulated genes of Ctrl_LW and ECM_LW samples of ST profile. d Spatial feature plot showing the distribution of IFEB 1 and IFED 1 subclusters in tissue sections. e Spatial feature plot showing the distribution of PF 1 and RF 1 subclusters in tissue sections. f Violin plots of FIB scores of individual spots derived from scRNA-seq data (sc-FIB score) for each subcluster. Dotted boxes stressed clusters with the higher average sc-FIB scores. g The spatial feature plot highlighted the expression of Krt28 + hair follicle progenitor and Twist2 + dermal condensate in migrating neo-epidermis. h Illustration showing the epithelialization along with de novo HF formation in the biomaterials-mediated healing process. i Spatial feature plot showing the distribution of MAC subclusters in tissue sections. j Violin plots of MAC scores of individual spots derived from scRNA-seq data (sc-MAC score) for each subcluster. Dotted boxes stressed clusters with the highest average sc-MAC score. k Representative IF images of stained AIM 1 (F4/80 + CD206 + ), white arrowheads showing the F4/80 + CD206 + cells. l Spatial feature plot and violin plot showing TC 1 distribution and expression level in tissue sections. m Spatial feature plot and violin plot showing the distribution and expression level of Treg 1 in tissue sections. n Representative IF images of stained Treg 1 (FOXP3), white arrowheads showing the FOXP3 + cells.

Article Snippet: For the evaluation the infiltration of immune cells, immunohistochemistry staining for CD3 (14-0032-82, Thermo Fisher Scientific, 1:100), CD68 (ab283654, Abcam, 1:100), Ly6G (ab238132, Abcam, 1:2000) and immunofluorescent staining for F4/80 (29414-1-AP, Proteintech, 1:200), CD206 (360017, Zenbio, 1:100), FOXP3 (sc-53876, Santa Cruz Biotechnology, 1:100), were performed.

Techniques: Derivative Assay, Expressing, Staining

Journal: Nature Communications

Article Title: Tracing immune cells around biomaterials with spatial anchors during large-scale wound regeneration

doi: 10.1038/s41467-023-41608-9

Figure Lengend Snippet: a The anatomical structure of each sample. b Gene enrichment analysis between WT and Rag2 -/- group. c Spatial feature plot and violin plot showing the distribution and expression level of integrated PF 2 subcluster in tissue sections. d Spatial feature plot and violin plot showing the distribution and expression level of Crabp1 (marker gene of PF 2 ) in tissue sections; Representative IF images of stained PF 2 (CRABP1 + ), white arrowheads showing the CRABP1 + cells. e Spatial feature plot and violin plot showing the distribution and expression level of integrated RF 2 subcluster in tissue sections. f Spatial feature plot and violin plot showing the distribution and expression level of Mest (marker gene of RF 2 ) in tissue sections; Representative IF images of stained RF 2 (MEST + ), white arrowheads showing the MEST + cells. g Spatial feature plot and violin plot showing the distribution and expression level of integrated AIM 2 subcluster in tissue sections. h Spatial feature plot and violin plot showing the distribution and expression level of Mrc1 (marker gene of AIM 2 ) in tissue sections; Representative IF images of stained AIM 2 (F4/80 + CD206 + ), white arrowheads showing the F4/80 + CD206 + cells. i Spatial feature plot and violin plot showing the distribution and expression level of integrated Treg 2 subcluster in tissue sections. j Spatial feature plot and violin plot showing the distribution and expression level of Foxp3 (marker gene of Treg 2 ) in tissue sections; Representative IF images of stained Treg 2 (FOXP3 + ), white arrowheads showing the FOXP3 + cells.

Article Snippet: For the evaluation the infiltration of immune cells, immunohistochemistry staining for CD3 (14-0032-82, Thermo Fisher Scientific, 1:100), CD68 (ab283654, Abcam, 1:100), Ly6G (ab238132, Abcam, 1:2000) and immunofluorescent staining for F4/80 (29414-1-AP, Proteintech, 1:200), CD206 (360017, Zenbio, 1:100), FOXP3 (sc-53876, Santa Cruz Biotechnology, 1:100), were performed.

Techniques: Expressing, Marker, Staining

Journal: PLoS Pathogens

Article Title: IL-22 and IDO1 Affect Immunity and Tolerance to Murine and Human Vaginal Candidiasis

doi: 10.1371/journal.ppat.1003486

Figure Lengend Snippet: ( A ) IDO1 protein and ( B ) gene expression in the vagina of C57BL/6 mice ( n = 4) intravaginally infected with C. albicans . Proteins in vaginal cell lysates (3 dpi) were visualized by western blotting with rabbit polyclonal IDO1 specific antibody. Scanning densitometry was done on a Scion Image apparatus. Western blots out of 2 independent experiments and corresponding pixel density ratio normalized against β-tubulin. Ido1 mRNA expression [normalized to mRNA of naïve (dpi 0) mice] in vaginal tissue (RT-PCR) at different dpi. ( C ) Relative concentrations of kynurenines (Kyn) and ( D ) kynurenine-to-tryptophan (Kyn/Trp) ratio in vaginal fluids at different dpi. Pooled results from 3 different experiments. * P <0.05, IDO1-deficient vs. C57BL/6 mice at the days indicated. N.S., not significant. ( E ) Vaginal fungal growth (Log 10 CFU/100 µl VF ± s.e.m.) at different dpi in mice ( n = 6) treated intraperitoneally with a mixture of l -kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid or PBS (None). Pooled data from 3 different experiments.* P <0.05, treated vs. untreated mice at the days indicated. N.S., not significant. ( F ) Periodic acid-Schiff-stained vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) acquired with a 40× and 100× objective, respectively, at 21 dpi. Scale bars, 100 µm. Representative image from 3 experiments. ( G ) Cytokine levels (pg/mg, cytokine/total proteins for each sample, at 21 dpi) in the vaginal fluids of mice treated as above. Pooled data from 3 different experiments. * P <0.05, treated vs. untreated (None) mice. ( H ) Vaginal immunohistochemistry of naïve or infected mice at 3 days after re-challenge. Double staining was done with anti-IL-10-FITC and polyclonal rabbit to FoxP3 followed by anti-rabbit TRITC. Cell nuclei were stained with DAPI (blue). Representative pictures (out of 2 experiments) were taken with a 20× objective. Scale bars, 50 µm.

Article Snippet: Double staining with FITC anti-IL-10 (JES5-16E3) and rabbit polyclonal to FOXP3 (abcam) was followed by anti-rabbit TRITC.

Techniques: Expressing, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Immunohistochemistry, Double Staining

Journal: Scientific reports

Article Title: Niraparib activates interferon signaling and potentiates anti-PD-1 antibody efficacy in tumor models.

doi: 10.1038/s41598-019-38534-6

Figure Lengend Snippet: Figure 2. Niraparib promoted tumor immune cell infiltration in both the BRCA-proficient SK6005 syngeneic and BRCA-deficient MDA-MB-436 NOG-EXL humanized tumor models (A) Representative images of CD4 and CD8 immunohistochemical staining in control- and niraparib-treated BRCA-proficient SK6005 tumors. (B–D) Quantification of the number of CD4+ cells, CD8+ cells and FoxP3+ cells per field upon niraparib treatment in BRCA-proficient SK6005 tumors. (E-G) Percentage of Ki67-positive CD4+ cells, CD8+ cells and FoxP3+ cells among the total CD3+ population by flow cytometry in humanized NOG-EXL MDA-MB-436 tumors. **p-value is less than 0.05 by Student’s t test.

Article Snippet: Immunohistochemical staining was performed with primary antibodies specific for CD4 (Sino Bio 50134-R001), CD8 (Affymetrix 14–0808), and FoxP3 (Novus NB100–39002) on the Bond RX system (Leica Biosystems, Germany).

Techniques: Immunohistochemical staining, Staining, Control, Flow Cytometry