Exosomes obtained from HepG2 cells incubated with DiI-LDL were absorbed onto 4 μm aldehyde-sulfate latex beads (Invitrogen, Life Technologies Corporation) and incubated first with CD63 antibody (1/200, Ref 55619, BD Biosciences) and then with a fluorophore-conjugated secondary antibody. Cells were washed and analyzed by flow cytometry (FACSscan, BD Biosciences) as previously described [39 (link)].
4 μm aldehyde sulfate latex beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 4 μm aldehyde/sulfate latex beads are a type of microscopic particles used in various laboratory applications. They have a diameter of 4 micrometers and possess both aldehyde and sulfate functional groups on their surface. These beads serve as a versatile tool for researchers and scientists working in fields such as biology, chemistry, and materials science.
Automatically generated - may contain errors
Lab products found in correlation
Cd63 antibody, by BD (1 mentions)
Facsscan, by BD (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Ab1318, by Abcam (1 mentions)
Ab79559, by Abcam (1 mentions)
Ab187372, by Abcam (1 mentions)
Alexa 488 tagged secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Cytexpert, by Beckman Coulter (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Anti cd63 apc antibody, by BioLegend (1 mentions)
Facscanto, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Spiroepoxide, by Santa Cruz Biotechnology (1 mentions)
Flojo software, by FlowJo (1 mentions)
Anti cd9 antibody, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Exoelisa ultra complete kit, by System Biosciences (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
11 protocols using 4 μm aldehyde sulfate latex beads
1
Exosome detection and quantification
2
Exosome Capture and Characterization
3
Exosome Quantification via CD9 Labeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exosomes were attached to 4 μm aldehyde/sulfate latex beads (Invitrogen, USA) by mixing 30 µg exosomes with 10 µL beads for 15 min at room temperature with continuous rotation. Subsequently, the bead-exosomes suspension was diluted to 1 mL PBS and rotated at room temperature for 30 min. The reaction was stopped by adding 100 mM glycine and 2% bovine serum albumin (BSA) (Beyotime, China) in PBS, followed by rotating at room temperature for 30 min. Exosomes-bound beads were then washed once in 2% BSA and centrifuged for 1 min at 10,000 rpm. Beads were blocked with 10% BSA at room temperature for 30 min with rotation, followed by a second wash in 2% BSA and centrifugation for 1 min at 10,000 rpm. Subsequently, the beads were incubated with an Alexa-488-tagged anti-CD9 antibody (312104, Biolegend, USA) for 30 min at 4 °C with rotation. After incubation, the beads were centrifuged for 1 min at 10,000 rpm, the supernatant was discarded, and the beads were washed in 2% BSA and centrifuged for 1 min at 10,000 rpm. The percentage of positive beads (those bound to CD9-positive exosomes) was calculated relative to the total number of beads analyzed per sample (10,000 events).
4
Characterization of EV Surface Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of CD9 and EpCAM on EVs were analyzed by flow cytometry as previously described (21 (link)). Briefly, EVs attached to 4 μm aldehyde/sulfate latex beads (Invitrogen, Carlsbad, CA, USA) were incubated with anti-CD9 antibodies (SAB4700092; Sigma-Aldrich, St. Louis, MO), anti-CD63 antibodies (ab1318; Abcam, Cambridge, MA, USA), anti-CD81 antibodies (ab79559; Abcam, Cambridge, MA, USA), or anti-EpCAM antibodies (ab187372; Abcam, Cambridge, MA, USA) for 30 min with rotation at 4°C followed by Alexa-488-tagged secondary antibodies (Life Technologies, Carlsbad, CA, USA) for 30 min with rotation at 4°C. Samples were detected using CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using CytExpert (Beckman Coulter, Brea, CA, USA).
5
Serum Exosome Identification by FACS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescent-activated cell sorting analysis of the serum exosomes was performed, as described previously.32 (link) Briefly, the exosomes isolated from the serum were conjugated with 4 μm aldehyde/sulfate latex beads (Invitrogen Life Technologies), followed by staining with phycoerythrin mouse antihuman CD63 or isotype control (BD Biosciences, San Jose, CA, USA). The analysis was performed using a BD FACScan flow cytometer (BD Biosciences).
6
Flow Cytometry Analysis of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A flow cytometry analysis of EVs was performed as described previously, with modifications [48 (link)]. Briefly, EVs, IL4RPep-1-FITC-EVs (without DOPE-BAM) or IL4RPep-1-FITC-EVs (with DOPE-BAM), were attached to 4 μm aldehyde/sulfate latex beads (Invitrogen) by mixing 5 μg of EVs in 10 μL of beads for 15 min at 37 °C. PBS was added to the above mixture to make a final volume of 1 mL, followed by incubation for 2 h on a rotating wheel at 37 °C. After incubation, the reaction was terminated by the addition of 100 mM glycine and centrifuged at 15,000× g for 2 min. The supernatant was discarded, and the beads were resuspended in 1 mL of PBS for flow cytometry analysis. The samples were analyzed using an automated high-speed cell counter/sorter (Beckman Coulter, Indianapolis, IN, USA).
7
Quantifying sEV-associated CD63 by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aliquots of 20 μg of sEVs were attached to 10 μl of 4 μm aldehyde/sulfate latex beads (Invitrogen) as described previously (24 (link)) with minor modifications. Briefly, to stop the reaction, sEVs-bound beads were treated with 100 mM glycine and 2% bovine serum albumin (BSA) in PBS and were blocked with 10% BSA and incubated with anti-CD63-APC antibody (BioLegend, #353007). Beads alone and beads + sEVs without antibody and beads+sEVs+isotype control (APC igG1, ƙ isotype Ctrl (FC) antibody (BioLegend, #400121) were used as controls to gate the beads with CD63-bound sEVs. The median fluorescence intensity of CD63 (∼100,000 events) was recorded in FACS Canto (BD Biosciences) and results were analyzed using the FlowJo software (BD Biosciences). Experiments were done in three replicates of each conditions.
8
Immuno-labeling of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MAC-EVs were made to attach to 4 μm aldehyde/sulfate latex beads (Invitrogen, CA, USA) by mixing 5 μg of MAC-EVs with a 10 μL volume of beads for 15 min. PBS was added to a final volume of 1 mL and the mixture was incubated in a rotary shaker for 2 h at room temperature. The reaction was stopped by adding 100 mM glycine and 2% BSA dissolved in PBS, and the mixture was again incubated in a rotary shaker for 30 min at room temperature. For every 10 μL of the MAC-EV-bound beads, 10 μL of Wnt3a and Wnt7b antibody mixture was incubated overnight at 4 °C, after which the mixture was centrifuged at 15,000× g for 2 min. The resulting supernatant was discarded. Alexa Fluor Fluorescein isothiocyanate (FITC) anti-rabbit antibody with 2% BSA dissolved in PBS was added to the mixture, which was then incubated at 37 °C for 60 min. Samples were diluted to 1 mL with 2% BSA in PBS and centrifuged for 2 min at 15,000× g. The supernatant was discarded, and the beads were resuspended in 1 mL PBS for flow cytometric analysis performed using a BD FACS Aria III instrument (BD Biosciences, NJ, USA).
9
Semiquantitative EV Detection In Vitro
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For semiquantitative detection of EVs in vitro, anti-mouse CD63 antibodies (Y-18, Santa Cruz Biotechnology) were coupled to 4 μm aldehyde/sulfate latex beads (Invitrogen) by incubating 35 μg of v with 1 × 108 beads, followed by blocking of remaining activated groups with 4% BSA in PBS44 (link).
In all, 2 × 105 MC38 tumour cells were seeded per well into 24-well plates in RPMI 1640 with 10% FCS in the presence of 0, 5 and 10 μM spiroepoxide, an inhibitor of neutral sphingomyelinase 2 (Santa Cruz Biotechnology). Twenty-four hours later, these supernatants were collected. Cell culture supernatants were filtered through 0.22-μm filters. Filtrates (100 μl) were incubated with 20,000 anti-CD63-coupled beads overnight at room temperature with gentle shaking. Beads were washed and incubated with phycoerythrin-anti-CD9 (MZ3, BioLegend, San Diego, CA, USA) for 30 min on ice. After washing in 2% BSA in PBS, the beads were analysed using FACS.
For inhibition of EV production in vivo, on day 0, 2-month-old mice were intraperitoneally injected with spiroepoxide in 200 μl PBS (2 g kg−1 body weight per mouse) or 200 μl of 3.75% DMSO PBS control every 48 h for 12 days (six injections total). Mice were killed 24 h after the final injection.
In all, 2 × 105 MC38 tumour cells were seeded per well into 24-well plates in RPMI 1640 with 10% FCS in the presence of 0, 5 and 10 μM spiroepoxide, an inhibitor of neutral sphingomyelinase 2 (Santa Cruz Biotechnology). Twenty-four hours later, these supernatants were collected. Cell culture supernatants were filtered through 0.22-μm filters. Filtrates (100 μl) were incubated with 20,000 anti-CD63-coupled beads overnight at room temperature with gentle shaking. Beads were washed and incubated with phycoerythrin-anti-CD9 (MZ3, BioLegend, San Diego, CA, USA) for 30 min on ice. After washing in 2% BSA in PBS, the beads were analysed using FACS.
For inhibition of EV production in vivo, on day 0, 2-month-old mice were intraperitoneally injected with spiroepoxide in 200 μl PBS (2 g kg−1 body weight per mouse) or 200 μl of 3.75% DMSO PBS control every 48 h for 12 days (six injections total). Mice were killed 24 h after the final injection.
10
Exosomal Surface Marker Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze the expression of exosomal surface markers, 4 μm aldehyde/sulfate latex beads (Thermo Fisher Scientific) were coated with anti-CD9 antibody (BD Biosciences, San Diego, CA; Cat: 555370) overnight and incubated with 30 μg of exosomes. The exosome-beads complexes were probed with human anti-CD81-PE (BD Biosciences; Cat: 555676) or human anti-CD63-PE (BD Biosciences; Cat: 557305) and data was acquired on a LSR II Flow cytometer (BD Biosciences). Data analysis was performed using the FloJo software (FlowJo version 10, Ashland, OR).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!