Journal: STAR Protocols

Article Title: Isolation, culture, and use of primary murine myoblasts in small-molecule screens

doi: 10.1016/j.xpro.2023.102149

Figure Lengend Snippet:

Article Snippet: bFGF Reconstitution Buffer 2 , R and D Systems , RB02.

Techniques: Plasmid Preparation, Recombinant, Modification, Cell Culture, Viability Assay, Software, Cytometry, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

doi: 10.1371/journal.pone.0049516

Figure Lengend Snippet: Differentiated PC-12, LA1-55n, Neuro-2a, and SiMa cells were treated (0.005–300 pM BoNT/A) for 24 h followed by 48 h incubation in toxin-free medium to allow for SNAP25 cleavage. A. Cell lysates were evaluated in a SNAP25 197 -WB and the data fitted to a 4PL model (SigmaPlot v10.0). The EC 50 = 6.5±0.9 pM (SEM) for SiMa cells while the EC 50 for the other cell lines could not be calculated. B. Summary table containing EC 50 and signal to background (S/B) at 1.2 and 300 pM for all the candidate cell lines. Historical data utilizing optimized dose-response curves for other cell lines is presented in parenthesis for comparison. C. Half of the cell lysates from A were tested in the newly developed ECL-sandwich ELISA shown in D. The EC 50 = 3.3±0.2 pM for SiMa cells while the other two cell lines that reached an upper asymptote produced EC 50 values of 58 pM (LA1-55n) and 54 pM (Neuro-2a). D. Format of the ECL-sandwich ELISA: capture with anti-SNAP25 197 monoclonal 2E2A6/detection with anti-SNAP25 polyclonal antibody S9684 (Sigma).

Article Snippet: For the ECL sandwich ELISA, MSD High Bind plates (Meso Scale Discovery) pre-spotted with anti-SNAP25 197 MAb 2E2A6 were blocked with 150 μL blocking buffer for 1 h at RT.

Techniques: Incubation, Comparison, Sandwich ELISA, Produced

Journal: PLoS ONE

Article Title: Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

doi: 10.1371/journal.pone.0049516

Figure Lengend Snippet: A. Differentiated SiMa cells were treated with 1 nM BoNT/A complex from 1 to 60 min. Cell lysates were evaluated in the ECL-sandwich ELISA. Signal above background was observed at the earliest time points indicating high affinity binding (Error bars = std. dev.). B. Specificity of uptake by SiMa cells was demonstrated by comparing the uptake of BoNT/A (150 kDa) to recombinant LH N /A (lacking binding domain but comprising the translocation domain and an active light chain) and inactive BoNT/A (iBoNT/A, inactive light chain). No cleavage of SNAP25 was detected with iBoNT/A. Non-specific uptake of LH N /A was observed only at doses >10 nM. C. Graph comparing specific uptake of BoNT/A complex (at pM concentrations) to a full dose-response of recombinant LH N /A (at pM to µM concentrations) with a highest dose of 50 µM. The EC 50 for the LH N /A molecule was 2.1 µM versus 0.85 pM for the fully active BoNT/A.

Article Snippet: For the ECL sandwich ELISA, MSD High Bind plates (Meso Scale Discovery) pre-spotted with anti-SNAP25 197 MAb 2E2A6 were blocked with 150 μL blocking buffer for 1 h at RT.

Techniques: Sandwich ELISA, Binding Assay, Recombinant, Translocation Assay

Journal: PLoS ONE

Article Title: Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

doi: 10.1371/journal.pone.0049516

Figure Lengend Snippet: A. Optimization of differentiation medium. SiMa cells were plated in RPMI-1640 with FBS (SM), RPMI-1640 serum-free medium (SFM) supplemented with N2 and B27, RPMI-1640 SFM with N2, BSA and NGF, or EMEM SFM with N2 and B27 for three days. Cells were treated with BoNT/A at 0.2, 2 and 20 pM for 24 h and incubated for 48 h. Lysates were analyzed by WB and the percent cleavage at 2 pM is shown. The same lysates were analyzed in the ECL-ELISA confirming that the EMEM SFM with N2 and B27 supplements was optimal for differentiation (Error bars = std. dev.). Clear signal over background was detected at 0.2 pM. B. Optimization of differentiation time. SiMa cells were differentiated for 6 to 72 h in EMEM SFM with GT1b, N2 and B27. Cells were treated with BoNT/A from 0.03 to 25 pM for 24 h followed by media change and 48 h incubation. The ECL-ELISA demonstrated that SiMa cells' sensitivity improved with ≥48 h differentiation. C. Optimization of BoNT/A treatment conditions . Differentiated SiMa cells were treated with 0.1 to 25 pM BoNT/A for 6 h or 24 h followed by incubation in toxin-free medium for 0, 16, 24, 48, or 72 h. Lysates were analyzed in the ECL-sandwich ELISA. EC 50 values were similar under all treatment conditions tested but S/B values were different. Optimal BoNT/A treatment to generate a low EC 50 and high S/B was 24 h followed by 2 or 3 days incubation. D. Optimization of the ECL-sandwich ELISA conditions. Concentrations of capture (2E2A6) and detection (S9684) antibodies were optimized. Capture antibody at 20 and 40 µg/mL was tested in combination with detection antibody at 1 and 5 µg/mL to analyze lysates from cells treated with BoNT/A from 0.01 to 25 pM. 2E2A6 at 20 µg/mL spotted in 5 µL combined with S9684 at 5 µg/mL in 25 µL was optimal (Error bars = std. dev.). E. Cell lysate incubation time and temperature were evaluated and optimal condition was 16 h incubation at 4°C.

Article Snippet: For the ECL sandwich ELISA, MSD High Bind plates (Meso Scale Discovery) pre-spotted with anti-SNAP25 197 MAb 2E2A6 were blocked with 150 μL blocking buffer for 1 h at RT.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Sandwich ELISA

Journal: PLoS ONE

Article Title: Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

doi: 10.1371/journal.pone.0049516

Figure Lengend Snippet: A. Protocols for the sensitive and screening (in italic) cell-based potency assays (CBPAs) utilizing differentiated SiMa cells and ECL-sandwich ELISA. B. Representative screening CBPA with SiMa cells treated with 0.014–10 nM BoNT/A (150 kDa) for 6 h followed by overnight incubation in toxin-free medium to allow for SNAP25 197 accumulation. Average EC 50 of 125 independent assays performed by three operators is shown. C. Dilutional linearity-recovery table with concentrations from 1.75 to 0.5 relative potencies (R.P.). The last column exemplifies how many individual assays produced data in which the confidence interval (C.I.) overlapped 1 making that dilution indistinguishable from the reference 1× preparation in that specific assay. Percent recoveries from 86 to 117% demonstrate excellent accuracy of the assay.

Article Snippet: For the ECL sandwich ELISA, MSD High Bind plates (Meso Scale Discovery) pre-spotted with anti-SNAP25 197 MAb 2E2A6 were blocked with 150 μL blocking buffer for 1 h at RT.

Techniques: Sandwich ELISA, Incubation, Produced

Journal: PLoS ONE

Article Title: Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

doi: 10.1371/journal.pone.0049516

Figure Lengend Snippet: A. Example of a representative experiment in which differentiated SiMa cells were treated with BoNT/A at concentrations from 0.03 to 25 pM for 24 h followed by 48 h incubation, and the lysates were evaluated in the optimized chemiluminescence read-out. The results obtained were similar to the ECL read-out (EC 50 ∼0.5 to 2 pM), excellent signal to background, and reproducibility of the replicates. B. Summary of optimized parameters for the chemiluminescence CBPA. Seven parameters comprising cell culture and ELISA read-out were specifically optimized for this assay by testing several conditions for each parameter.

Article Snippet: For the ECL sandwich ELISA, MSD High Bind plates (Meso Scale Discovery) pre-spotted with anti-SNAP25 197 MAb 2E2A6 were blocked with 150 μL blocking buffer for 1 h at RT.

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay