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: 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: Nature Communications

Article Title: GPR4 promotes immune exclusion in colon cancer through LOXL2-mediated extracellular matrix remodeling

doi: 10.1038/s41467-025-67967-z

Figure Lengend Snippet: A GPR4 regulates JAK2 and STAT3 phosphorylation via western blotting. n = 3. Scale bar: 100 µm. B Representative IF images of STAT3-Y705 phosphorylation regulated by GPR4. C Quantitative analysis of STAT3-Y705 phosphorylation in GPR4 - OE SW480 cell line and ( D ) GPR4 - KD HCT116 cell lline. Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. E Static treatment reduces both pSTAT3-Y705 and the expression of LOXL2, COL1A1, and TGF-β in GPR4 - OE SW480 cells. F RT-PCR analysis for LOXL2 in SW480 cell lines after receiving Stattic treatment. Two-tailed t test, n = 3 biologically independent samples. G RT-PCR analysis for TGF-β in SW480 cell lines after receiving Stattic treatment. Data are presented as mean ± SD.Two-tailed t test, n = 3 biologically independent samples. H STAT3 transcription factor binding site. I In SW480 cells, STAT3 binds to the LOXL2 promoter region confirmed via ChIP assays. J STAT3 binds to the TGF-β promoter region confirmed via ChIP assays. K RT-PCR assesses the transcriptional activity of LOXL2 promoter regions and TGF-β promoter region L . Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. ns > 0.05.

Article Snippet: Subsequently, Transcription Factor Staining Kit (Elabscience, E-CK-A108) was applied for cells fixation and Permeabilization as recommended method.

Techniques: Phospho-proteomics, Western Blot, Two Tailed Test, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay

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

Journal: Biomedicines

Article Title: Clinicopathological Features of Gastric Cancer with Autoimmune Gastritis

doi: 10.3390/biomedicines10040884

Figure Lengend Snippet: Mucin type, immune cell numbers, PD-L1 expression, and bacteria.

Article Snippet: For immunofluorescence, the paraffin-embedded sections were incubated with primary antibodies against MUC2 (cat. no. ab90007; Abcam, Cambridge, UK), MUC5AC (cat. no. ab3649; Abcam), MUC6 (cat. no. NBP2-44374; Novusbio, Littleton, CO, USA), CD3 (cat. no. NCL-L-CD3-565; Leika, Wetzlar, Germany), CD11b (cat. no. 14011282; Invitrogen, Waltham, MA, USA), Foxp3 (cat. no. 98377; Cell Signaling Technology), PD1 (cat. no. 86163; Cell Signaling Technology), Bacillus cereus (cat. no. ab20556; Abcam), and Streptococcus Group B (cat. no. ab53584; Abcam).

Techniques: Expressing, Bacteria

Journal: Biomedicines

Article Title: Clinicopathological Features of Gastric Cancer with Autoimmune Gastritis

doi: 10.3390/biomedicines10040884

Figure Lengend Snippet: Immunohistochemical staining for PD-L1, CD3, CD11b, Foxp3, and PD1 in gastric cancer in pure AIG and H. pylori /APCA-negative patients. Elevated PD-L1 expression and infiltration of CD3/Foxp3/PD1-positive cells in gastric cancer is associated with pure AIG. Scale bars, 200 μm.

Article Snippet: For immunofluorescence, the paraffin-embedded sections were incubated with primary antibodies against MUC2 (cat. no. ab90007; Abcam, Cambridge, UK), MUC5AC (cat. no. ab3649; Abcam), MUC6 (cat. no. NBP2-44374; Novusbio, Littleton, CO, USA), CD3 (cat. no. NCL-L-CD3-565; Leika, Wetzlar, Germany), CD11b (cat. no. 14011282; Invitrogen, Waltham, MA, USA), Foxp3 (cat. no. 98377; Cell Signaling Technology), PD1 (cat. no. 86163; Cell Signaling Technology), Bacillus cereus (cat. no. ab20556; Abcam), and Streptococcus Group B (cat. no. ab53584; Abcam).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: PLoS ONE

Article Title: Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function

doi: 10.1371/journal.pone.0173773

Figure Lengend Snippet: There was no difference in GFR of patients with predominantly methylated (>75% Foxp3 TSDR methylation) Treg and patients with a sizable proportion of demethylated (≤75% Foxp3 TSDR methylation) Treg (p>0.01; Mann-Whitney U test). Enriched IFNy+ and IFNy- Treg of male and female patients were tested separately using primer ADS783 (primer P1) and primer ADS3576 (primer P2).

Article Snippet: PCR was carried out in a 21 μl total volume containing: 2.1 μl PCR buffer, 0.27 μl MgCl 2 , 0.42 μl dNTPs, 1.05 μl SYBR Green dye (EpigenDx), 1.26 μl each of primers ADS783 and ADS3576 (Human Foxp3 Methylation Panel, EpigenDx), 0.12 μl Hot Start Taq polymerase (Qiagen), and 1.5 μl of bisulfite-treated genomic DNA (concentration 5 ng/μl).

Techniques: Methylation, MANN-WHITNEY