Normalized Median Fluorescence: An Alternative Flow Cytometry Analysis Method for Tracking Human Embryonic Stem Cell States During Differentiation

Chan, Lesley Y.; Yim, Evelyn K.F.; Choo, Andre

doi:10.1089/ten.tec.2012.0150

Cited by 36 publications

(29 citation statements)

References 20 publications

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“…In addition to measuring the overall surface expression and SEB binding, the three clones with the highest SEB : SAPE binding ratio were analyzed by flow cytometry for percent binding to (percentage of cells outside of the gated, unlabeled control population for each clone) and median fluorescence intensity of (Chan et al ., ) four proteins for specificity analysis: NAPE, SAPE, PA‐488, and α HA‐FITC. There was minimal binding observed for the three clones at concentrations less than 1000 nM; therefore, the on‐cell specificity analysis was conducted using 1000 nM of each protein, a concentration almost 7‐fold greater than the 150 nM used for SEB (Figure ; Supplemental Figures 4, 5).…”

Section: Resultsmentioning

confidence: 99%

Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting

Kogot

Pennington

Sarkes

et al. 2014

J of Molecular Recognition

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Bacterial peptide display libraries enable the rapid and efficient selection of peptides that have high affinity and selectivity toward their targets. Using a 15-mer random library on the outer surface of Escherichia coli (E.coli), high-affinity peptides were selected against a staphylococcal enterotoxin B (SEB) protein after four rounds of biopanning. On-cell screening analysis of affinity and specificity were measured by flow cytometry and directly compared to the synthetic peptide, off-cell, using peptide-ELISA. DNA sequencing of the positive clones after four rounds of microfluidic magnetic sorting (MMS) revealed a common consensus sequence of (S/T)CH(Y/F)W for the SEB-binding peptides R338, R418, and R445. The consensus sequence in these bacterial display peptides has similar amino acid characteristics with SEB peptide sequences isolated from phage display. The Kd measured by peptide-ELISA off-cell was 2.4 nM for R418 and 3.0 nM for R445. The bacterial peptide display methodology using the semiautomated MMS resulted in the discovery of selective peptides with affinity for a food safety and defense threat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Journal of Molecular Recognition published by John Wiley & Sons, Ltd.

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Section: Resultsmentioning

confidence: 99%

Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting

Kogot

Pennington

Sarkes

et al. 2014

J of Molecular Recognition

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“…The voltage settings on the LSRII may vary between experiments. In order to have a reproducible MFI value for PD-L1 expression on all tumor cell lines, we therefore normalized the MFI values for all experiments by making a ratio between the MFI for stained cells divided by the MFI for control stained cells (27). In addition, each human tumor cell line was scored on a quartile scale (ranging from 1 to 4) for both % PD-L1 positive cells and PD-L1 MFI.…”

Section: Methodsmentioning

confidence: 99%

Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti–PD-L1 Antibody Avelumab (MSB0010718C) on Human Tumor Cells

Boyerinas

Jochéms

Fantini

et al. 2015

Cancer Immunology Research

398

350

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Several anti-PD1/PD-L1 monoclonal antibodies (MAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these MAbs is to inhibit PD1 on immune cells interacting with PD-L1 on tumor cells. These MAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective MAb-mediated cancer therapies. A fully human anti-PD-L1 MAb would potentially be able to block PD-L1/PD1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 MAb. The studies reported here demonstrate (a) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (b) IFNγ can enhance tumor cell PD-L1 expression and in some cases enhance ADCC tumor cell lysis; (c) purified NK cells are potent effectors for avelumab; (d) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (e) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (f) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 MAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity.

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“…1). 26,27,36,[39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56] Mohr et al generated various sizes of EBs from hESCs using different sizes of microwells. 26 Subsequently, the EBs were reseeded and cultured in EB media in gelatin-coated dishes.…”

Section: Hpsc Differentiation In Type Bmentioning

confidence: 99%

Physical cues of cell culture materials lead the direction of differentiation lineages of pluripotent stem cells

et al. 2015

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Both human pluripotent stem cells (hPSCs) from embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential ability to differentiate into many different cell types originating from all three germ layers. This review discusses physical cues from natural and synthetic biomaterials that guide the differentiation of hESCs and hiPSCs into several different lineages. We place special focus on how hPSC differentiation fate is affected by (a) the elasticity of biomaterials used for hPSC culture, (b) the topography of biomaterials used for hPSC culture, and (c) the mechanical forces associated with biomaterials (stretching and electrical stimulation via biomaterials) used for hPSC culture.

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Normalized Median Fluorescence: An Alternative Flow Cytometry Analysis Method for Tracking Human Embryonic Stem Cell States During Differentiation

Cited by 36 publications

References 20 publications

Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting

Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting

Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti–PD-L1 Antibody Avelumab (MSB0010718C) on Human Tumor Cells

Physical cues of cell culture materials lead the direction of differentiation lineages of pluripotent stem cells

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