high-binding flat bottom 384-well plates  (Greiner Bio)

Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: a Image depicting amino acids in the TEM8 MIDAS motif that coordinate the metal ion (green). b ELISA was used to measure the binding of AP, TEM8-AP, and D150A-AP to col1. PA was included as a positive control. n = 2 (AP, negative control) or 3 (TEM8-AP and TEM8-D150-AP) biologically independent samples per group. Statistical comparison between AP and D150A- AP was performed using an unpaired T test. c ELISA was used to measure the binding of AP and D150A-AP to various ECM molecules. PA was included as a positive control. n = 3 biologically independent samples per group. Statistical comparison between AP and D150A-AP was performed using an unpaired T test. d IF staining was used to detect TEM8 (green) in CHO and CHO-TEM8 cells. Bar = 20 μm. Images were representative of three experiments. e A cell binding assay was used to measure binding of CHO-TEM8 cells to various ECM molecules. n = 6 biologically independent samples per group. Statistical comparisons between CHO and CHO-TEM8 were performed using an unpaired T test. f IF staining was used to detect CHO-mediated degradation of an underlying FITC-col gel (green). Cell nuclei were visualized using DAPI (blue). Bar = 50 μm. Images were representative of three experiments. g IF staining was used to detect collagen uptake after adding soluble FITC collagen (green) to the media. CellMask orange was used to visualize cell membranes (red) and Hoechst 33342 to visualize nuclei (blue). Bar = 20 μm. Images were representative of three experiments. h Western blotting was used to detect changes in TEM8 expression following exposure of CHO-TEM8 cells to various ECM molecules. Wedge: Col1; 10, 25, and 50 μg/mL, ColVI; 1, 10, 25 μg/mL. Images were representative of three experiments. Data in b , c , and e , are denoted as mean ± s.e.m. Source data are provided as a file.

Article Snippet: Stock solutions of 1 mg/mL rat tail collagen were diluted to a 2× final concentration in 0.1% acetic acid, then solid phased onto UltraCruz High Binding ELISA Multiwell Microplates (Santa Cruz Biotechnology) by diluting to desired concentration (i.e., 200 μg/mL) in 50 mM Tris-HCl (pH 8). pH neutralization allows fibrillar matrix formation and attachment to wells .

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Positive Control, Negative Control, Comparison, Staining, Cell Binding Assay, Western Blot, Expressing

Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: a Alignment of TEM8 with the known collagen-binding site of integrins. The conserved mutations in this study, including the Glutamine (E) and Histidine (H) found in collagen binding integrins are highlighted (yellow). b Image depicting the conserved TEM8 surface residues, E152 and H154, predicted to contact collagen based on homology with integrin alpha 2. c An ELISA was used to measure the binding of AP, AP-TEM8 (WT) and various AP-TEM8- mutants to col1. n ≥ 8 biologically independent samples per group. Statistical analysis was calculated using one-way analysis of variance with a Tukey’s test. d A FITC release assay was used to measure soluble FITC in the supernatant of CHO cells, CHO cells expressing wildtype TEM8 (WT) or various TEM8 mutants following culture in a FITC-Col gel. n = 4 biologically independent samples per group. P < 0.0001 between CHO- TEM8 WT and each of the CHO-TEM8 mutants. Statistical analysis was calculated by using one-way analysis of variance with a Tukey’s test. e Flow cytometry was used to measure FITC in CHO, CHO-TEM8 (WT), and CHO-TEM8 mutant cells following FITC-Col treatment. f Growth of MC38 in TEM8 +/+ or TEM8 E150V/E150V mice. n = 20 (TEM8 +/+ ) and 14 (TEM8 E150V/E150V ) biologically independent animals per group. Mice used in this study were 4 to 5 months old. Statistical analysis was calculated using unpaired T tests comparing tumor volume from WT and KI mice on the same day post inoculation. *; P < 0.0001, **; P = 0.002, ***; P = 0.009. Data are denoted as mean ± s.e.m. Source data are provided as a file.

Article Snippet: Stock solutions of 1 mg/mL rat tail collagen were diluted to a 2× final concentration in 0.1% acetic acid, then solid phased onto UltraCruz High Binding ELISA Multiwell Microplates (Santa Cruz Biotechnology) by diluting to desired concentration (i.e., 200 μg/mL) in 50 mM Tris-HCl (pH 8). pH neutralization allows fibrillar matrix formation and attachment to wells .

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Release Assay, Expressing, Flow Cytometry, Mutagenesis

Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: a ELISA was used to measure the binding of AP (control) and TEM8-AP to col1 in the absence or presence of 20 μg/mL of m830 anti-TEM8 antibodies. n = 11 biologically independent samples per group. Statistical analysis was calculated by using an unpaired T test. b Flow cytometry was used to quantify FITC-Col uptake in CHO and CHO-TEM8 cells that were treated with non-specific control IgG (IgG) or TEM8 antibodies m830 or SB5. The SB5 anit-TEM8 antibody is unable to block collagen binding and was used as an additional negative control. c Growth of UACC melanoma tumors following treatment with 15 mg/kg of m830 antibody. Treatments were administered 3× per week and initiated (arrow) when tumors reached a size of 60 mm 3 . n = 18 (vehicle) or 12 (m830) biologically independent animals per group. *; P ≤ 0.0001, **; P = 0.0002. Statistical analysis was calculated using unpaired T tests comparing tumor volume from vehicle and m830 treated mice on the same day post inoculation. d Average body weights of mice in c at treatment start (day 6) and study end (day 28). N.S.: non-significant. n = 15 (vehicle) or 12 (m830) independent samples per group. Statistical analysis was calculated by using an unpaired T test. Liver metastases following intrasplenic injection of HCT-116-luc colon cancer was quantified at 21- and 28-days post inoculation (DPI) ( e ) using BLI. Statistical analysis was calculated by using an unpaired T test. Images from five representative mice/group are shown in f and examples of excised livers with tumor lesions (arrowheads) are shown in g . Kaplan- Meier survival analysis ( h ). In this study, treatments began with 15 mg/kg one day following tumor cell inoculation (arrow) followed by 5 mg/kg 3 times/week for 4 weeks. Log-rank analysis: P < 0.0001, m830 versus vehicle. n = 15/group. i Growth of DLD1 colon tumors following treatment with 15 mg/kg of m830 3× per week, 5 mg/kg bevacizumab (Bev) 2× per week, or a combination of m830 and Bev. Treatments were initiated (arrow) when tumors reached an average size of 50 mm 3 . n = 18 (vehicle), 13 (m830), 13 (Bev), or 14 (m830 + Bev) biologically independent animals per group. Statistical analysis was calculated by using an unpaired T test. j Growth of NCI-H460 lung tumors following treatment with 15 mg/kg of m830 3x per week, 30 mg/kg paclitaxel (PT) (qod × 5), or a combination of m830 and PT. Treatments were initiated (arrow) when tumors reached an average size of 60 mm 3 . n = 10 biologically independent animals per group. Statistical analysis was calculated by using an unpaired T test. k Growth of MC38 colon tumors following treatment with 10 mg/kg of m830, 3.5 mg/kg anti- PD1 antibody (αPD1, clone RMPI), or a combination of m830 and αPD1. Treatments were administered 3× per week and were initiated (arrow) when tumors reached an average size of 80 mm 3 . p = 0.01 with respect to αPD1 alone. n = 12 (vehicle), 10 (m830), 14 (anti-PD1) or 14 (m830 + anti-PD1) biologically independent animals per group. Statistical analysis was calculated by using an unpaired T test. Data in a and d are denoted as mean ± s.d. Data in c, i–k are denoted as mean ± s.e.m. Source data are provided as a file.

Article Snippet: Stock solutions of 1 mg/mL rat tail collagen were diluted to a 2× final concentration in 0.1% acetic acid, then solid phased onto UltraCruz High Binding ELISA Multiwell Microplates (Santa Cruz Biotechnology) by diluting to desired concentration (i.e., 200 μg/mL) in 50 mM Tris-HCl (pH 8). pH neutralization allows fibrillar matrix formation and attachment to wells .

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Flow Cytometry, Blocking Assay, Negative Control, Injection

Journal: Journal of Controlled Release

Article Title: Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine

doi: 10.1016/j.jconrel.2022.05.023

Figure Lengend Snippet: MPLA+PUUC NPs increase T cell responses in the lung when delivered intranasally with spike protein. On days 0 (1st dose) and 28 (2nd dose), female BALB/c mice were immunized I.N. with unformulated (i.e., soluble) or NP-conjugated spike protein (1 μg) and PLGA-PEI NPs (4 mg) loaded with MPLA (24 μg), PUUC (17 μg), and MPLA+PUUC (20 μg, 17 μg). Mice were euthanized and lungs were collected on day 35, one week after the 2nd dose. Lung cells were restimulated with spike peptide pools for 6 h and stained for analysis by flow cytometry. Percentages of cells expressing A) CD4 + CD44 + out of CD45 + cells, B) IFNγ + out of CD4 + CD44 + cells, C) TNFα + out of CD4 + CD44 + cells, D) CD69 + CD103 + (tissue resident memory T cells) out of CD45 + cells. BAL fluid from vaccinated mice using soluble spike antigen was assayed for anti-spike E) IgG and F) IgA with ELISA. G) Sera were assayed for anti-spike IgG with ELISA (error bars represent the SEM). p values are * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 calculated using A – D) One-way ANOVA with Tukey post-hoc test, E , F) Kruskal-Wallis with Dunn's post-hoc test for nonparametric data, or G) Two-way ANOVA with Tukey post-hoc test.

Article Snippet: Spike-neutralizing antibodies were quantified using a modified ELISA assay in a 384-well UltraCruz® ELISA high-binding plate (Santa Cruz Biotechnology).

Techniques: Staining, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Controlled Release

Article Title: Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine

doi: 10.1016/j.jconrel.2022.05.023

Figure Lengend Snippet: PUUC NPs delivered intramuscularly with spike protein enhance humoral responses. Female BALB/c mice were immunized I.M. into both tibialis anterior muscles at day 0 (1st dose) with soluble spike protein at doses of 80 ng, 200 ng, 1000 ng with or without adjuvant-NPs (4 mg) loaded with PUUC (+P, 20 ng PUUC dose). Peripheral blood was sampled on day 26. On day 28, mice received a 2nd dose of protein subunit vaccines. Mice received the same formulations, except for two groups that received 80 ng spike protein as a 1st dose received 1000 ng spike protein as a 2nd antigen dose (80/1000 and 80/1000 +P). Mice were euthanized on day 36 for to collect blood and popliteal LNs. A) Anti-spike IgG in post-1st dose sera at various dilutions measured by absorbance at 450 nm during ELISA assays and B) comparison of area under the curve (AUC). C – D) Anti-spike IgG in post-2nd dose sera measured by absorbance at 450 nm and comparison of AUC. E) ACE-2 signal measured by absorbance at 450 nm in spike protein neutralization assay with post-2nd dose sera. Absorbance was normalized to a blank well in each row of a 384 well plate to correct for plate effects. Lower absorbance values indicate higher spike-neutralizing antibody levels in sera. Percentages of cells expressing F) Bcl6 + out of B220 + cells, G) GL7 + out of B220 + cells and H) CXCR5 + out of B220 − cells from combined popliteal lymph nodes. B,D) Normality was assessed with the Kolmogorov-Smirnov test. Statistical significance was determined with the Kruskal-Wallis test and Dunn's post-hoc test for multiple comparisons. E – H) Statistical significance calculated with One-Way ANOVA and Tukey post-hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 for all graphs.

Article Snippet: Spike-neutralizing antibodies were quantified using a modified ELISA assay in a 384-well UltraCruz® ELISA high-binding plate (Santa Cruz Biotechnology).

Techniques: Muscles, Adjuvant, Vaccines, Enzyme-linked Immunosorbent Assay, Comparison, Neutralization, Expressing