The ABD‐engineered EVs (1×1011 dose) and the corresponding control EVs were incubated with FITC labelled HSA (HSA‐FITC, ab8030, Abcam) and the final concentration of HSA‐FITC was 150 μg/ml at 37°C for 120 min. And then the EVs were subjected to size exclusions by using the IZON qEV columns (IZON, qEVoriginal/70 nm). 48 fractions were collected from each sample with 300 μl per fraction. 150 μl of each fraction was taken in 96‐well plate for fluorescence intensity detection by using SpectraMax i3x (MOLECULAR DEVICES, USA). 30 μl of each fraction was taken for luciferases (Nanoluc or Thermoluc) activities assay by using GloMax 96 Microplate Luminometer machine (Promega, USA).
Glomax 96 microplate luminometer machine
Manufactured by Promega
Sourced in United States, Sweden
The GloMax 96 Microplate Luminometer is a compact and sensitive instrument designed for the detection of luminescent signals in 96-well microplates. It utilizes a photomultiplier tube to measure the intensity of light emissions, allowing for the quantification of various luminescent assays, such as luciferase reporter gene assays, ATP detection, and more.
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6 protocols using glomax 96 microplate luminometer machine
1
HSA-FITC Labeling and Quantification of EVs
2
Nanoluc-Based EV/Tissue Detection
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Determined amount of EVs (such as 1×109) or 10 μl of tissue lysate from mice internal organs were used for Nanoluc detection. The EVs or tissue lysate were added into white‐walled 96‐well plates. 25 μl diluted Nano‐Glo substrates (Nano‐Glo Luciferase Assay System: Promega), were added into each well by injection mode of the GloMax 96 Microplate Luminometer machine (Promega, USA). The background wells without any nanoluc proteins were also included for future calculations.
3
Luciferase Assay for Protein Quantification
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Luciferase assay protocol reported by Saher et al. [32 (link)] was followed with some modifications. Cells were washed with 1× PBS and then lysed with 25 µL of 0.1% Triton-X100 reagent. 5 µL of cell lysates were used to determine protein quantity using the DC Protein Assay (Bio-Rad, Hercules, CA, USA) protocol. The remaining 20 µL of the lysates were mixed with luciferase reagent (Promega, Stockholm, Sweden) automatically by the injector. The RLU of luciferase were determined (GloMax® 96 Microplate Luminometer machine-Promega, Stockholm, Sweden). The final results were represented as fold increase in luciferase activity, calculated by normalizing the RLU values to the total protein quantities and then further normalization against values of untreated cells.
4
Luciferase Assay with Protein Normalization
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A modification of the previously reported protocol for the luciferase assay was followed [41 (link)]. The medium was removed, and the cells lysed in 25 μL of 0.1% Triton-X 100 in 1X PBS per well in a 96-well plate. After lysis of the cells, 5 μL were used to determine total protein quantity by DC Protein Assay (BioRad). The remaining 20 μL of lysates were mixed with 50 μL of the luciferase reagent (Promega) added via injector. The relative light units (RLU) of luciferase were measured (GloMax® 96 Microplate Luminometer machine, Promega, Sweden) with 10 s integration time and 2 s delay between injection and measurement. The values were divided by the total protein quantities determined and normalized to untreated well values. Final results are represented as fold increase in luciferase activity.
5
Quantifying siRNA Knockdown Efficiency
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HeLa_Luc705 and HuH7_Luc705 cells were seeded in 96-well plates at 10,000 cells per well in growth media with 1 µM or 100 nM Luc705-SSO. SMCs were diluted directly into prepared growth media. After 24 h to allow adherence and SSO internalization, 10 µl of diluted SMC was added into each well at indicated concentrations. For positive control, SSOs were complexed with Lipofectamine 2000 for 30 min at RT and cells treated with a final concentration of 200 nM. ON concentrations above 200 nM would require LF2000 to be used in excess of the cells toxicity threshold. After treatment, media was removed from the wells and replaced with fresh growth media for 4 h to allow translation of luciferase. The media was then removed again and cells lysed with 0.1% Triton X-100 (Sigma Aldrich, cat no. X-100) in 1× PBS (Gibco, cat no. 10010023).
For the detection of luciferase activity, 30 μL of cell lysate was transferred to white-walled 96-well plates (Sigma Aldrich, cat no. CLS3922). The luciferase intensity in each well was immediately measured (n = 3, N = 3) using a GloMax® 96 Microplate Luminometer machine (Promega) following auto-injection of 25 μL Luciferin substrate per well as per the Promega Firefly Luciferase Assay System (Thermo Scientific, cat no. 16174). The exposure time of photon measurements was set to 10 s, delayed 2 s after injection.
For the detection of luciferase activity, 30 μL of cell lysate was transferred to white-walled 96-well plates (Sigma Aldrich, cat no. CLS3922). The luciferase intensity in each well was immediately measured (n = 3, N = 3) using a GloMax® 96 Microplate Luminometer machine (Promega) following auto-injection of 25 μL Luciferin substrate per well as per the Promega Firefly Luciferase Assay System (Thermo Scientific, cat no. 16174). The exposure time of photon measurements was set to 10 s, delayed 2 s after injection.
6
EV Uptake in B16F10 and TCMK-1 Cells
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B16F10 and TCMK‐1 cells were seeded in 24‐well plates and incubated at 37°C overnight. 1×109 EVs from different groups were added directly into the cells the second day. After 2 h, 4 h and 8 h incubation of the EVs with recipient cells, the cell supernatants were removed and washed with 1 ml PBS twice and then the cells were lysed with 100 μl 0.1% TritonX‐100, 15 min in a shaking machine (900 rmp/min). 30 μl of the cell lysates were took for Nluc activity assay by using GloMax 96 Microplate Luminometer machine (Promega, USA).
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