The frozen naïve MSCs and eMSCs-IL10 were thawed for this assay. An MSC Phenotyping Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, catalog# 130-125-285) was used to detect MSC surface molecule expression according to the manufacturer’s protocols. The antibody cocktail consists of CD73, CD90, CD105, CD45, CD34, CD19, CD14, and HLA-DR. A MACSQuant Analyzer 16 flow cytometer was used to collect the data. A MACS® Comp Bead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, catalog# 130-104-187) was used for compensation before each run.
Macs comp bead kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The MACS Comp Bead kit is a laboratory equipment product designed to perform compensation setup for flow cytometry analysis. It contains fluorescently labeled beads that can be used to determine the appropriate compensation settings for multicolor flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Msc phenotyping kit, by Miltenyi Biotec (1 mentions)
Facsaria 3, by BD (1 mentions)
Propidium iodide, by Miltenyi Biotec (1 mentions)
Macsquant running buffer, by Miltenyi Biotec (1 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Cd45 fitc, by Miltenyi Biotec (1 mentions)
Ca9 pe, by Miltenyi Biotec (1 mentions)
Cd326 pe vio770, by Miltenyi Biotec (1 mentions)
Cd105 pe vio770, by Miltenyi Biotec (1 mentions)
Facs buffer, by Miltenyi Biotec (1 mentions)
Macsquant analyzer 10 flow cytometer, by Miltenyi Biotec (1 mentions)
5 protocols using macs comp bead kit
1
Characterization of Mesenchymal Stem Cells
2
Multiparameter Flow Cytometry Profiling of CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Surface marker staining was performed on enriched CD4+ T cells from PBMCs at day zero, CD4+ T cells co-culture with sMSCs, nsMSCs (direct contact and Transwell system), sHUV-EC-C cells, nsHUV-EC-C cells (direct contact and Transwell system) and CD4+ T cells cultured alone at day ten. Cells were pelleted down and resuspended in 100 μl staining buffer (containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA). Antibodies CD4-VioGreen (0.034 μg/ml), CCR10-PE (0.034 μg/ml), CD183 (CXCR3)-PE-Vio770 (0.02 μg/ml), CD194 (CCR4)-APC (0.068 μg/ml), CD196 (CCR6)-PE-Vivo-615 (0.013 μg/ml) were added and incubated for 10 min at 4 °C in the dark. Cells were washed with 1 ml staining buffer, pelleted and resuspended in 100 μL MACSQuant running buffer (Miltenyi Biotec). 5 μL of Propidium iodide (Miltenyi Biotec) was added shortly before running the samples on FACSARIA III (BD Biosciences). Data were analyzed with FlowJo software (v10, FlowJo LLC). To compensate optimally for fluorescence spillover from fluorochrome-conjugated antibodies, the MACS Comp Bead Kit, anti-human (130-104-187, Miltenyi Biotec) and anti-mouse (130-097-900, Miltenyi Biotec) Igκ(Miltenyi Biotec) were used.
3
Comprehensive Immune Cell Phenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This section describes the methods for the phenotyping shown in Fig. 2 . All samples were washed in FACS buffer (1× PBS, 2% FCS), stained for a minimum of 30 min at 4 °C before additional washes and running flow cytometry on the MACSQuant Analyzer 10 (Miltenyi Biotec). The MACS Comp bead kit (catalog no. 130-097-900; Miltenyi Biotec) was used for compensation controls. The antibodies used for these experiments are listed in Supplementary Table 2 .
4
Multiparameter Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Digested tumor or adjacent normal cells were centrifuged and resuspended at 5 × 105–2 × 106 cells/100 µL FACs buffer (Miltenyi, Bergisch Gladbach, Germany). Cells were divided into no antibody control, viability analysis panel (Propidium iodide, Hoeschst33342, DRAQ7), IgG control (REA-VioBlue, REA-FITC, REA-PE, REA-APC, REA-PE-Vio770), staining panel 1 (1:50 CD31-VioBlue (Miltenyi 130-117-227), 1:11 CA9-PE (Miltenyi 130-110-057), 1:10 PDGFRβ-APC (Miltenyi 130-105-322), 1:10 PDGFRα-APC (Miltenyi 130-115-239), 1:50 CD326-PE-Vio770 (Miltenyi 130-111-002), 1:50 CD45-FITC (Miltenyi 130-110-631), Propidium Iodide), or staining panel 2 (1:50 CD10-VioBlue (Miltenyi 130-114-509), 1:11 CA9-PE, 1:50 CD105-PE-Vio770 (Miltenyi 130-112-167), CD184-APC (Miltenyi 130-098-357), 1:50 CD326-PE-Vio770, Propidium Iodide), and incubated in the dark at 4 °C for 15 min. Cells were washed two times with flow buffer and analyzed using Miltenyi AutoMACs flow cytometer. Side and forward scatter gating were determined using viability analysis panel to identify cells vs. debris. Doublets were excluded using SSR-H versus SSR-L. Positive gating for each marker was determined using IgG control. Compensation was performed using the MACS Comp Bead kit, anti-REA (Miltenyi 130-104-693).
5
Flow Cytometric Analysis of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to analysis of stained samples by flow cytometry, compensation settings were determined using single color controls and unstained cells. Single color controls were prepared using the MACS Comp Bead kit (Miltenyi #130-104-693). Cells were washed and stained in PBS containing 2% bovine serum albumin (BSA). Prior to flow-cytometric analysis of PBMC, staining was performed using a panel of monoclonal antibodies and CAR Detection Reagents (Supplementary Table 1 ) following manufacturer’s instructions. Costaining of cytoplasmic markers was performed following fixation and permeabilization, using Inside Fix and Inside Perm from the SARS-CoV-2 T Cell Analysis Kit (Miltenyi #130–128-034). Live cells were identified by 7-AAD dye exclusion (Miltenyi #130–111-568). Samples were acquired using a Miltenyi MACSQuant Analyzer 10 Flow Cytometer.
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!