We screened mouse surface markers using the “Mouse cell surface marker screening panel” Lyoplate (BD Biosciences, material number 562208) according to the manufacturer’s instructions. In brief, 150×10^6 cells were used for our partially reprogrammed cells (passage 15) and Oct4-neoR mES cells. Cells were plated on 0.2% gelatin coated plates and treated with neomycin three days before screening. 175×10^6 cells were used for passage 4 Sox2-EGFP MEFs. Cells were washed with PBS-EDTA, dissociated in 10× TrypLE (Invitrogen) for 5 minutes, washed once in PBS before staining for 30 minutes in primary antibody in staining medium (PBS-EDTA supplemented with 0.5% BSA) according to manufacturer’s instructions, washing once in PBS, staining for 30 minutes in fluorophore conjugated secondary antibody in staining medium, washing once in PBS and analyzed on a BD LSR Fortessa FACS analyzer in in staining medium. We used a high concentration of TrypLE to verify that all identified markers would not be cleaved by our dissociation reagent. We further validated a subset of these identified makers with our control populations dissociated in 1× TrypLE and stained with fluorophore conjugated antibodies as described below.
Lyoplate
Manufactured by BD
Lyoplate is a laboratory equipment designed for freeze-drying applications. It provides a controlled environment for the lyophilization process, which is used to preserve and stabilize various biological samples, such as proteins, cells, and pharmaceuticals.
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7 protocols using lyoplate
1
Mouse Cell Surface Marker Screening
2
Cell Surface Marker Profiling in DIPG
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Cell surface markers present on DIPG cell cultures were screened using a panel of monoclonal antibodies against human cell surface markers (Lyoplate, BD Biosciences). Low passage (<12) DIPG cultures expanded from tumor tissue collected at autopsy in serum-free, neurosphere forming conditions40 were allotted to 96 well plates and blocked with 1ug of goat IgG per million cells to reduce nonspecific binding of secondary antibodies subsequently used in the assay. Cells were then incubated sequentially with primary and secondary antibodies with intermediate wash steps according to the manufacturer’s instructions. Dead cells were then labeled with a Live/Dead violet stain (ThermoFisher), and following washes cells were fixed in 1% PFA for 10 minutes at room temperature. The following day, stained cells were analyzed by flow cytometry. Doublets and dead cells were excluded by gating, and the mean fluorescence intensity of antibody labeling for each target on the panel was normalized to the mean fluorescence intensity for the matched isotype control per the manufacturer’s recommendations.
3
Cell Surface Marker Profiling in DIPG
4
Comprehensive Immunophenotyping of Cell Populations
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A total of 242 primary antibodies (Lyoplate, BD Biosciences) were stained with Alexa 647-conjugated secondary antibodies and costained with anti-CD271 PE (clone C40-1457). Data were confirmed by serial repetition of positives and controlled by known negative antibodies. Population boundary limits were based on (1) cells only for evaluation of autofluorescence and setting of PMTs, (2) isotypic control for anti-CD271 (same isotype, concentration, and fluorochrome), (3) secondary antibody only (rat anti-mouse IgG or goat anti-rat IgG Alexa 647), (4) FMO to set boundaries and check compensation for CD271, and (5) anti-CD271 plus secondary antibody to ascertain any nonspecific Alexa 647 background on p75NTR+ cells. A marker was considered “positive” when expressed by at least 35% of the cells and “upregulated” when the fold change (FC) was ≥2.
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Mouse Cell Surface Marker Screening
6
Surface Receptor Profiling of Monocyte-T Cell Co-Cultures
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CD14+ monocytes and CD4 T cells were isolated from PBMCs, differentiated, co-cultured for 48 h, and CD4 T cells were sorted as described above. After sorting, the surface receptors of CD4 T cells were stained with the BD Lyoplate (BD) consisting of a panel of 242 monoclonal primary antibodies and Alexa Flour 647-conjugated goat anti-mouse IgG and goat anti-rat IgG secondary antibodies, and subsequently analyzed by flow cytometry. In parallel, M2 cultures were analyzed with the same antibody panel. Figure 1 A was generated using the ggplot2 package.
7
Screening Cell Surface Markers of Chemically Treated Cells
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SCRC-4000 cells (5 × 105) were plated onto φ60 mm culture dishes and were cultured for 2 days (day 0–2). Then, cells were culture for 6 days with (chem sample) or without (control sample) small molecules (days 2–8). On day 7, cells were collected by enzymatic dissociation with TrypLE Express and were re-plated onto 96-well plates at 1 × 105 cells/well. The next day (day 8), chem and control samples were stained with 242 antibodies from human cell surface marker screening panel (BD Lyoplate). Fluorescence was observed and analyzed as described above.
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