The largest database of trusted experimental protocols

3 protocols using flow cytometry fixation permeabilization buffer kit 1

1

Dissociation and Profiling of HIO Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
HIOs were removed from Matrigel and washed 3-4 times in phosphate-buffered saline and subsequently incubated in TrypLE Express (Life Technologies, Camarillo, CA, USA) for 2-3 minutes until the organoids were completely disassociated to a single cell suspension. Single cells were then fixed and permeabilized using Flow Cytometry Fixation & Permeabilization Buffer Kit I (R&D Systems). Cells were then stained with the following fluorophore-conjugated antibodies: mouse anti-human EpCAM (# 324212; BioLegend, San Diego, CA, USA) and mouse anti-human vimentin (#562338; BD Pharmingen, San Diego, CA, USA). Cells were analyzed on a BD Fortessa Cell Analyzer (BD Biosciences) and flow cytometry analysis was carried out using FlowJo software (FlowJo, Ashland, OR, USA).
+ Open protocol
+ Expand
2

Tumor Immune Cell Profiling Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Tumor masses were cut into pieces and digested into mononuclear cell suspension according to the protocol 31 (link). Then, mononuclear cell suspension was blocked with anti- mouse CD16/32 (BD Biosciences, San Jose, CA, USA), followed by incubating at 4 °C for 30 min with fluorescein-conjugated specific antibodies against surface antigens CD45 (#563891, BD Pharmingen, CA, USA), CD11b (#553310, BD Pharmingen), F4/80 (#565411, BD Pharmingen), MHCII (#557000, BD Pharmingen), CD206 (FAB25351R-100UG, R&D Systems), CD3e (#551163, BD Pharmingen), CD4 (#552051, BD Pharmingen), and CD8a (#553030, BD Pharmingen). Then, intracellular staining was performed using Flow Cytometry Fixation & Permeabilization Buffer Kit I (#FC009, R&D Systems). IFN-γ (#554412, BD Pharmingen) and TNF-α (#554420, BD Pharmingen) antibodies were used for intracellular staining. Matched non-specific isotype immunoglobulins were stained as controls. 7-ADD attaining was used to exclude dead cells. After washing twice with staining buffer, cells were resuspended in 300 μl of PBS with 1% FBS and analyzed using a BD LSRFortessa flow cytometer (BD Biosciences). Results were processed and visualized with FlowJo V10 software (TreeStar, Ashland, OR, USA).
+ Open protocol
+ Expand
3

3D Matrigel Cell Dissociation and FACS Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
The cells grown in 3D matrigel were dissociated with dispase (17105041 ThermoFisher) at 37 °C for 1 hour centrifuged at 300 g × 5 min and washed in 1x cold PBS. To generate single cell suspensions, cell pellets were incubated in TrypLE for 10–15 minutes. The reaction was diluted in DMEM/F12 (Life Technologies) with 2% (v/v) FBS and centrifuged at 300 g at room temperature. The cell pellets were immersed with sorting buffer 3% (v/v) FBS in PBS, cell aggregates were removed using a cell strainer with a 70-µm pore size (BD falcon) and single cells were collected. For surface antigens, the cells were incubated with primary antibodies for 30 minutes with gentle shaking, washed twice with 3% (v/v) FBS/PBS, and if necessary, incubated with the secondary antibodies for 30 minutes followed by two more rinses with 3% (v/v) FBS/PBS. For intracellular antigens, the Flow Cytometry Fixation & Permeabilization Buffer Kit I, Flow Cytometry Permeabilization/Wash Buffer I and Flow Cytometry Staining Buffer kits (R&D). The cells were analyzed using a BD FACS Aria II flow cytometer (BD Biosciences). Unstained controls were used for gating conjugated antibodies and secondary only controls were used for gating unconjugated antibodies. The list of antibodies used for FACS is shown in Supplementary Table S2.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!