anti human anti adenosine a 2a r rabbit polyclonal antibody  (Alomone Labs)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeted knockdown of the adenosine A 2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells

doi: 10.1016/j.omtm.2021.03.001

Figure Lengend Snippet: ADORA2A siRNAs decrease ADORA2A mRNA and A 2A R protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.

Article Snippet: A 2A R expression post-transfection is as follows: activated HD CD3 + or CD8 + T cells transfected with scr or A 2A R siRNAs were fixed with 1%–4% paraformaldehyde (Affymetrix, Thermo Fisher Scientific), permeablized with BD Perm/Wash buffer (Fixation/Permeabilization Solution kit; BD Cytofix/Cytoperm Plus, BD Biosciences) and then stained with anti-human anti-adenosine A 2A R rabbit polyclonal antibody (Alomone labs) in BD Perm/Wash buffer followed by staining with a secondary anti-rabbit antibody—either Alexa Fluor-647 conjugated donkey anti-rabbit antibody (Thermo Fisher) or Brilliant Violet 421 conjugated donkey anti-rabbit antibody (BioLegend).

Techniques: Expressing, Labeling, Transfection, Standard Deviation, Flow Cytometry, Fluorescence, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeted knockdown of the adenosine A 2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells

doi: 10.1016/j.omtm.2021.03.001

Figure Lengend Snippet: CD45RO(A 2A R)-NPs rescue the chemotactic ability of HNSCC CD8 + memory T cells in the presence of adenosine (A and B) Representative two point trajectories of T cells migrating along the CXCL10 gradient (green) or CXCL10 + adenosine (ADO) gradient (blue) in HNSCC CD8 + memory T cells treated with (A) CD45RO(scr)-NPs or (B) CD45RO(A 2A R)-NPs. Trajectories artificially set to start at the origin with the red triangle representing the Y-COM. (C) Y-COM of HNSCC CD8 + memory T cells treated with (left) CD45RO(scr)-NPs (n = 6) or (right) CD45RO(A 2A R)-NPs (n = 6) in the presence of CXCL10 or CXCL10 + adenosine. Significance was determined using (left) a Wilcoxon signed rank test or (right) paired Student’s t test. (D) Percent inhibition of Y-COM of HNSCC CD8 + memory T cells in the presence of CXCL10 + adenosine compared to CXCL10 alone when treated with CD45RO(scr)-NPs (n = 6) compared to CD45RO(A 2A R)-NPs (n = 6). Data are represented single dot plots (colored dots; wherein each dot represents a single donor) and as mean ± standard deviation (shown in black). Significance was determined using Student’s t test.

Article Snippet: A 2A R expression post-transfection is as follows: activated HD CD3 + or CD8 + T cells transfected with scr or A 2A R siRNAs were fixed with 1%–4% paraformaldehyde (Affymetrix, Thermo Fisher Scientific), permeablized with BD Perm/Wash buffer (Fixation/Permeabilization Solution kit; BD Cytofix/Cytoperm Plus, BD Biosciences) and then stained with anti-human anti-adenosine A 2A R rabbit polyclonal antibody (Alomone labs) in BD Perm/Wash buffer followed by staining with a secondary anti-rabbit antibody—either Alexa Fluor-647 conjugated donkey anti-rabbit antibody (Thermo Fisher) or Brilliant Violet 421 conjugated donkey anti-rabbit antibody (BioLegend).

Techniques: Inhibition, Standard Deviation