After having withdrawn 350 µL of each sample, the rest of the ThinPrep PreservCyt solution was vortexed for 3×10 seconds with the swab tip inside the 20 mL vial. A 5 mL aliquot of the sample was centrifuged at 1,500 rpm for 5 minutes at RT. The cell pellet was resuspended in 100 µL of PBS (pH 7.4), supplemented with 2 mM EDTA and 0.5% BSA. Cells were fixed again for 20 minutes at RT with 100 µL of Inside Fix (MACS Inside Stain kit; Miltenyi Biotec, GmbH). Fixed cells were washed twice with PBS. The washed cells pellet was stained for 10 minutes at RT with 100 µL of anti-cytokeratin antibody (Miltenyi Biotec, GmbH) diluted with Inside Perm (MACS Inside Stain kit; Miltenyi Biotec, GmbH). The stained cells were then washed with Inside Perm (MACS Inside Stain kit; Miltenyi Biotec, GmbH). A second wash was performed with PBS supplemented with 2 mM EDTA and 0.5% BSA. The final washed pellet was resuspended in 500 µL PBS supplemented with 2 mM EDTA and 0.5% BSA. Samples were then analyzed by flow cytometry using Accuri C6 analyzer (Becton Dickinson, Franklin Lakes, NJ, USA).
Macs inside stain kit
Manufactured by Miltenyi Biotec
Sourced in Germany, Canada
The MACS Inside Stain Kit is a product designed for intracellular staining and flow cytometric analysis. It enables the detection of intracellular antigens within cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd68 fitc, by Miltenyi Biotec (1 mentions)
Cd163 pe, by Thermo Fisher Scientific (1 mentions)
Cd86 pe vio770, by Miltenyi Biotec (1 mentions)
Cd206 pe, by Thermo Fisher Scientific (1 mentions)
Cd11b vio bright fitc, by Miltenyi Biotec (1 mentions)
Fc500 flow cytometer, by Beckman Coulter (1 mentions)
Anti nkg2c, by R&D Systems (1 mentions)
Live dead fixable far red, by Thermo Fisher Scientific (1 mentions)
Anti tnf α mab11, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ 4s b3, by Thermo Fisher Scientific (1 mentions)
Moflo astrios eq flow cytometer, by Beckman Coulter (1 mentions)
Lyse buffer, by BD (1 mentions)
Lsrii cytometer, by BD (1 mentions)
4 protocols using macs inside stain kit
1
Cytokeratin-Based Cell Enrichment
2
Multicolor Flow Cytometry Analysis of Macrophage Phenotypes
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All data were acquired using a Cytomics FC 500 flow cytometer operating on CXP software equipped with a dual 488 nm/635 nm (blue/red) laser (Beckman Coulter, Brea, CA, USA). Intracellular staining and staining of cell surface antigens were performed simultaneously using the MACS Inside Stain Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, Inside Fix buffer was added to 1 × 105 cells and then resuspended in PBS. The solution was incubated at room temperature and pressure for twenty minutes, after which the cells were washed twice with PBS via centrifugation for five minutes at 300 g. The supernatant was discarded, and the pellet was resuspended in 100 µL of Inside Perm buffer; cells were stained with fluorochrome-conjugated antibodies at appropriate dilutions. Cells were incubated for fifteen minutes at room temperature and then washed with Inside Perm buffer. The supernatant was discarded, the pellet was resuspended in 400 µL of PBS, then flow cytometric analysis was performed. Antibodies against CD11b-VioBright FITC, CD86-PE-Vio770, and CD68-FITC were obtained from Miltenyi Biotec, (Bergisch Gladbach, Germany), whilst antibodies against CD206-PE and CD163-PE were obtained from eBioscience (San Diego, CA, USA).
3
PBMC Immunophenotyping Workflow
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PBMC were stained for extracellular markers (clones in parenthesis) with anti-CD2 (RPA-2.10, BioLegend, San Diego, CA, USA), anti-CD3 (BW264/56), anti-CD16 (3G8), anti-CD56 (REA196), anti-CD57 (TB03), anti-NKG2A (REA110), all from Miltenyi Biotec, Auburn, CA, USA and anti-NKG2C (134591, R&D Systems, Minneapolis, MN, USA). LIVE/DEAD Fixable Far Red (Invitrogen) was used to exclude dead cells. Fixation and permeabilization were performed using MACS Inside Stain Kit (Miltenyi Biotec) with polyclonal goat anti-human FcεRIγ (EMD Millipore, Etobicoke, ON, Canada), anti-IFN-γ (4S.B3), and anti-TNF-α (MAb11) from eBioscience, San Diego, CA, USA added for intracellular staining. The fluorescence minus one strategy was used to adjust multicolor compensation. Phenotypic analysis was performed using Kaluza Flow Cytometry Software 1.2 after data acquisition on a MoFlo Astrios EQ flow cytometer (both Beckman Coulter, Brea, CA, USA).
4
Flow Cytometry Analysis of Stromal Vascular Fraction
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