Hello,
I´m analyzing mesenchymal stem cells from fat in flow cytometry, but I need to assure that I am selecting the population of interest, and not adipocytes. We have used oil red in slices for this, but I have read that it can also be used in flow cytometry. Have someone a protocol for this?
Thank you all
Cristina
Some good references for you:
Article Novel flow cytometric approach for the detection of adipocyt...
https://www.jlr.org/article/S0022-2275(20)31815-0/fulltextLet me know if you have any questions ; )
Dear Cristina!
You also look at this article,please:
Assay validation for the assessment of adipogenesis of multipotential stromal cells—a direct comparison of four different methods
https://core.ac.uk/download/pdf/82542669.pdf
Mesenchymal stromal cells (MSCs)
Oil red staining for semiquantitative scoring of adipogenesis
Before staining, cells were washed twice with PBS and fixed in 10% (v/v) formalin (Biostain Ready Reagents, Manchester, UK). After fixation and two further washes in PBS, oil red solution [0.5% oil red (w/v) (Sigma) in isopropanol] was added for 10 min. After incubation at room temperature (RT), oil red was removed, and cells were washed twice in PBS and stained with Harris hematoxylin (Surgipath, Bretton, UK) for 45 seconds. Subsequently, cells were washed in tap water and allowed to dry before viewing. The amount of fat in individual cells was scored semiquantitatively through the use of a grading scheme developed by English et al. (25). The level of adipogenesis was described by ranking 500 cells in a middle area of the well on their fat content. Ranks were allocated on the basis of the proportion of cytoplasm occupied by fat globules: grade 1: 0e24%; grade 2: 25e49%; grade 3: 50e74%; grade 4: 75e100% (Figure 1A). Subsequently, a relative percentage of cells assigned to each grade was calculated for triplicate wells and averaged.
Flow cytometry for FABP4 and Nile red staining For Nile red staining, cells were harvested by treatment with 0.5% trypsin/EDTA (Invitrogen), washed in PBS and fixed with 10% (v/v) formalin in PBS. Stock Nile red (Sigma) solution (0.1 mg.mL1 , prepared in methanol) was added to cells at a final concentration of 0.05 mg mL1 (1:2000 dilution in PBS) (17) and incubated at 4C for 20 min. Cells were washed in BD FACSflow before flow cytometry was carried out with the use of a FACScan. Nile red fluorescence emission in adipogenically differentiated MSCs was detected on FL2 channel (bandpass filter, 585 42 nm). FABP4 protein was detected with the use of a purified polyclonal goat anti-human FABP4 antibody (IgG, R & D Systems, Abingdon, UK) at 1:20 dilution in PBS. For intracellular staining with FABP4, trypsinized cells were initially fixed in 100 mL of Intrastain reagent A (Dako, Cambridge, UK). FABP4 antibody was added together with 100 mL of permeabilizing reagent/Intrastain reagent B (Dako) for 15 min at RT in the dark. After two washes with PBS, cells were incubated with biotinilated rabbit anti-goat secondar antibody (1:400 dilution in PBS, Dako) for 15 min at RT in the dark. After two more washes in PBS, the binding was visualized with the use of streptavidinFITC (1:50 dilution, BD Sciences). After two final washes, the fluorescence was analyzed on FL1 channel (bandpass filter, 530 30 nm), with 10000 events being collected in each sample.
Flow cytometry for MSC marker expression The following antibodies were used to test MSC marker expression on undifferentiated and day-16 adipogenically differentiated BM MSCs (n ¼ 2) and skin fibroblasts (n ¼ 1): CD73-PE, CD90-PE, CD166-PE, CD44-PE (all from BD, Oxford, UK), CD105-PE, CD13-FITC, CD31-FITC (all from Serotec, Kidlington, UK) and CD45-FITC (Dako, Ely, UK). All isotype controls were from Serotec, and extended phenotyping of polyclonal and clonal cultures was performed as previously described (32).
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