picokinetm elisa catalog number ek0405 data analysis the average of the duplicate readings was obtained for every sample control  (Boster Bio)

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood–Brain Barrier

doi: 10.3390/ijms25179262

Figure Lengend Snippet: CD200 on brain endothelial cells. ( A ) Relative quantification shows higher CD200 mRNA abundance in Canx −/− or Fabp5 −/− cells compared to wild-type cells (based on 7 experiments performed in duplicates). ( B ) Representative histograms of negative control (dashed line), unstimulated cells (grey histogram), and cytokine stimulated cells (blue histogram). ( C ) CD200 + cells in wild-type, Canx −/− , and Fabp5 −/− bEND.3 cell populations. ( D ) Cell surface abundance (mean fluorescence intensity, MFI) of CD200 on unstimulated and cytokine-treated wild-type, Canx −/− , and Fabp5 −/− cells. ( E ) Percentage of CD200 in CD11b − PECAM-1 + endothelial cells isolated from brains of Canx −/− and wild-type mice. ( F ) MFI values of gated CD200 + CD11b − PECAM-1 + brain endothelial cells. All data shown are from 7 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: On the day of analysis, the samples were thawed at room temperature and mixed by vortexing, and 100 μL of each sample was used for the quantification of sCD200, using the mouse CD200 ELISA Kit PicokineTM (Boster Bio Cat#EK1185, Pleasanton, CA, USA) as per the manufacturer-recommended protocol.

Techniques: Quantitative Proteomics, Negative Control, Fluorescence, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood–Brain Barrier

doi: 10.3390/ijms25179262

Figure Lengend Snippet: Transmigration of activated T-cells across wild-type, Canx −/− , or Fabp5 −/− bEND.3 cell monolayers. ( A ) Percentage of activated T-cells migrated through Canx −/− or Fabp5 −/− bEND.3 cell monolayers vs. wild-type bEND.3 cell monolayers (white bars). The use of anti-CD200 blocking antibody increased T-cell transmigration across wild-type, Canx −/− , or Fabp5 −/− bEND.3 cell monolayers (blue bars), while the use of the control isotype antibody did not (grey bars). ( B ) Representative histograms of wild-type, Canx −/− , or Fabp5 −/− bEND.3 cells treated with CD200 siRNA and non-targeting siRNA. ( C ) The siRNA-mediated downregulation of CD200 in wild-type, Canx −/− , or Fabp5 −/− bEND.3 cell monolayers also increased the transmigration of T-cells across wild-type, Canx −/− , and Fabp5 −/− bEND.3 cell monolayers. Data representative of 8 independent experiments analyzed in triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: On the day of analysis, the samples were thawed at room temperature and mixed by vortexing, and 100 μL of each sample was used for the quantification of sCD200, using the mouse CD200 ELISA Kit PicokineTM (Boster Bio Cat#EK1185, Pleasanton, CA, USA) as per the manufacturer-recommended protocol.

Techniques: Transmigration Assay, Blocking Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood–Brain Barrier

doi: 10.3390/ijms25179262

Figure Lengend Snippet: CD200-dependent binding of T-cells to wild-type, Canx −/− , and Fabp5 −/− bEND.3 cells. ( A ) Relative quantification of T-cells adhered to wild-type, Canx −/− , and Fabp5 −/− bEND.3 cells. ( B ) Representative images of T-cells (green) adhered to wild-type, Canx −/− , and Fabp5 −/− bEND.3 cells (red). ( C ) Increased T-cell adherence to wild-type, Canx −/− , and Fabp5 −/− bEND.3 cells in the presence of anti-CD200 antibodies. * p = 0.0325, ** p = 0.006, *** p = 0.0011, **** p < 0.0001. Data shown are the combined results of 3 experiments performed in triplicates. Blue, DAPI staining.

Article Snippet: On the day of analysis, the samples were thawed at room temperature and mixed by vortexing, and 100 μL of each sample was used for the quantification of sCD200, using the mouse CD200 ELISA Kit PicokineTM (Boster Bio Cat#EK1185, Pleasanton, CA, USA) as per the manufacturer-recommended protocol.

Techniques: Binding Assay, Quantitative Proteomics, Staining

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood–Brain Barrier

doi: 10.3390/ijms25179262

Figure Lengend Snippet: Endothelial cell-derived soluble CD200. ( A ) Soluble CD200 (sCD200) in conditioned media of wild-type, Canx −/− , or Fabp5 −/− endothelial bEND.3 cells. (5 experiments in duplicates). ( B ) Abundance of sCD200 in serum collected from wild-type and Canx −/− mice. (n = 3 per group). ( C ) Representative images (n = 3 views per experiment) of T-cells (green) adhered to wild-type bEND.3 monolayers cultured in the absence or presence of serum collected from wild-type and Canx −/− mice. * p = 0.0325, ** p = 0.0057, **** p < 0.0001. The experiment was repeated 3 times in triplicates. Blue, DAPI staining ( D ) Ratio of T-cells to wild-type bEND.3 cells in cultures incubated with serum collected from wild-type (black dots) or Canx −/− mice (red symbols). ( E ) A schematic representation of how the binding of sCD200 produced by endothelial cells to CD200R1 on T-cells antagonizes the adhesion of T-cells with endothelial cells and thereby inhibits the process of transmigration. Wild-type endothelial cells of the blood–brain barrier (left panel) produce a basal amount of CD200 and sCD200. By contrast, Canx −/− or Fabp5 −/− brain endothelial cells produce elevated levels of CD200 and sCD200. The amount of sCD200 produced saturates the CD200R1 on activated T-cells, preventing them from interacting with endothelial cells, thus attenuating T-cell adhesion and transmigration across the blood–brain barrier (middle panel). The addition of excess anti-CD200 antibodies antagonizes the binding of CD200 and sCD200 produced by Canx −/− or Fabp5 −/− brain endothelial cells to CD200R1 on T-cells, thus enabling them to interact with CD200 on endothelial cells and subsequently cross the blood–brain barrier (right panel).

Article Snippet: On the day of analysis, the samples were thawed at room temperature and mixed by vortexing, and 100 μL of each sample was used for the quantification of sCD200, using the mouse CD200 ELISA Kit PicokineTM (Boster Bio Cat#EK1185, Pleasanton, CA, USA) as per the manufacturer-recommended protocol.

Techniques: Derivative Assay, Cell Culture, Staining, Incubation, Binding Assay, Produced, Transmigration Assay

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood–Brain Barrier

doi: 10.3390/ijms25179262

Figure Lengend Snippet: CD200 in brain endothelial cells from MS lesions. Acute MS brain lesions demonstrate low levels of CD200 (asterisk) and thus lack co-localization with PECAM-1 + brain endothelial cells ( A – C ). Unaffected brain white matter shows high CD200 abundance and co-localization with PECAM-1 + brain endothelial cells ( D – F ). Scale bar: 50 µm. Representative images of samples from 2 unaffected and 6 MS donors.

Article Snippet: On the day of analysis, the samples were thawed at room temperature and mixed by vortexing, and 100 μL of each sample was used for the quantification of sCD200, using the mouse CD200 ELISA Kit PicokineTM (Boster Bio Cat#EK1185, Pleasanton, CA, USA) as per the manufacturer-recommended protocol.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood–Brain Barrier

doi: 10.3390/ijms25179262

Figure Lengend Snippet: CD200 is lacking in MS lesions compared with unaffected white matter. Representative image of an MS lesion in white matter, left to the lesion border, adjacent to grey matter, right to the lesion border. ( A – D ) shows a juxtacortical lesion, a well-recognized type of lesion in MS. Samples were stained for CD200 (green) ( A , B , D , F ) and neuronal marker NeuN (red) ( C , E , F ). CD200 was not detected in the endothelial layer (open arrows in D , E and solid arrows in F ) in the blood vessels marked with an asterisk within the lesion, while neurons showed immunopositivity for CD200 outside the lesion area ( B ). Positive immunostaining for CD200 in unaffected brain blood capillaries (open arrows) and neuronal cell bodies (open arrowheads) ( A , D ). Scale bar: 100 µm for A , 50 µm for B – F . Representative images of samples from 2 unaffected and 6 MS donors.

Article Snippet: On the day of analysis, the samples were thawed at room temperature and mixed by vortexing, and 100 μL of each sample was used for the quantification of sCD200, using the mouse CD200 ELISA Kit PicokineTM (Boster Bio Cat#EK1185, Pleasanton, CA, USA) as per the manufacturer-recommended protocol.

Techniques: Staining, Marker, Immunostaining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Prenatal treatment with preimplantation factor improves early postnatal neurogenesis and cognitive impairments in a mouse model of Down syndrome

doi: 10.1007/s00018-024-05245-9

Figure Lengend Snippet: Treatment with sPIF restores DYRK1A and BDNF protein levels in the brain of Dp(16)1Yey pups on P6. Evaluation of the DYRK1A protein level in WT pups (vehicle: n = 5; sPIF: n = 5) and Dp(16)1Yey pups (vehicle: n = 3; sPIF: n = 5) ( A ). The brain BDNF protein concentration in WT pups (vehicle: n = 6; sPIF: n = 4) and Dp(16)1Yey pups (vehicle: n = 4; sPIF: n = 6) ( B ). Data are expressed as the mean ± SD and were analyzed in a two-way ANOVA followed by Fisher’s least squares difference test. ** p < 0.01

Article Snippet: The levels of brain-derived neurotrophic factor (BDNF) in brain lysates were measured with a mouse BDNF PicokineTM ELISA kit (Boster).

Techniques: Protein Concentration

Journal: Chemico-biological interactions

Article Title: Signature of paraoxonases in the altered redox homeostasis in Alzheimer's disease.

doi: 10.1016/j.cbi.2023.110839

Figure Lengend Snippet: Fig. 4. PON1 (panel A) and PON2 (panel B) protein levels in transgenic (3xTg-AD) and non-transgenic (Non-Tg; i.e. controls) mice brain homogenate at different post-natal days (PD). Data are reported as mean ± S.E.M.; (n = 4/group). Statistical analysis was performed by 2-way ANOVA and subsequent Bonferroni post-hoc test for multiple comparisons. *p < 0.05, **p < 0.01.

Article Snippet: PON1 and PON2 proteins concentration in homogenized brains were determined by PicokineTM Mouse PON1 ELISA kit (Boster Biochem, Cambridge, MA, USA) and mouse PON2 ELISA kit (Abcam, Cambridge, UK) following the manufactured procedures.

Techniques: Transgenic Assay