An improved procedure for isolating adult mouse cardiomyocytes for epicardial activation mapping.

Cardiovascular disease is a leading cause of death and disability worldwide. Although genetically modified mouse models offer great potential for robust research in vivo, in vitro studies using isolated cardiomyocytes also provide an important approach for investigating the mechanisms underlying cardiovascular disease pathogenesis and drug actions. Currently, isolation of mouse adult cardiomyocytes often relies on aortic retrograde intubation under a stereoscopic microscope, which poses considerable technical barriers and requires extensive training. Although a simplified, Langendorff-free method has been used to isolate viable cardiomyocytes from the adult mouse heart, the system requires enzymatic digestions and continuous manual technical operation. This study established an optimized approach that allows isolation of adult mouse cardiomyocytes and epicardial activation mapping of mouse hearts using a Langendorff device. We used retrograde puncture through the abdominal aorta in vivo and enzymatic digestion on the Langendorff perfusion device to isolate adult mouse cardiomyocytes without using a microscope. The yields of isolated cardiomyocytes were amenable to patch clamp techniques. Furthermore, this approach allowed epicardial activation mapping. We used a novel, simplified method to isolate viable cardiomyocytes from adult mouse hearts and to map epicardial activation. This novel approach could be beneficial in more extensive research in the cardiac field.

Studying the microglia response to oxidized phosphatidylcholine in primary mouse neuron culture and mouse spinal cord.

Oxidized phosphatidylcholine (OxPC) found in multiple sclerosis brain lesions mediates neurodegeneration. Microglia are prominent responders to the OxPC insult, and thus, studying their protective or noxious functions is important to help halt neurodegeneration. Here, we present protocols including cell isolation and culture, animal surgeries, as well as tissue processing and isolation to study the microglia response to OxPC-mediated neurodegeneration in vitro and in vivo. For complete details on the use and execution of this protocol, please refer to Dong et al. (2021).

Magnetic-Activated Cell Sorting Using Coiled-Coil Peptides: An Alternative Strategy for Isolating Cells with High Efficiency and Specificity.

Magnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers. Current MACS systems generally require an antigen to be expressed at the cell surface; these antigen-presenting cells subsequently interact with antibody-labeled magnetic particles, facilitating separation. Here, we present an alternative MACS method based on coiled-coil peptide interactions. We demonstrate that HeLa, CHO, and NIH3T3 cells can either incorporate a lipid-modified coiled-coil-forming peptide into their membrane, or that the cells can be transfected with a plasmid containing a gene encoding a coiled-coil-forming peptide. Iron oxide particles are functionalized with the complementary peptide and, upon incubation with the cells, labeled cells are facilely separated from nonlabeled populations. In addition, the resulting cells and particles can be treated with trypsin to facilitate detachment of the cells from the particles. Therefore, our new MACS method promotes efficient cell sorting of different cell lines, without the need for antigen presentation, and enables simple detachment of the magnetic particles from cells after the sorting process. Such a system can be applied to rapidly developing, sensitive research areas, such as the separation of genetically modified cells from their unmodified counterparts.

Development of decellularized aortic scaffold for regenerative medicine using Sapindus mukorossi fruit pericarp extract.

The aim of this study is to develop a novel decellularization method using aqueous extract of soap nut pericarp (SPE) and its evaluation using hematoxylin-eosin staining, scanning electron microscopy, diamidino-2-phenylindol (DAPI) staining, mechanical testing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and DNA quantification. The presently available decellularization agent raises some concerns due to the potential for presence of residual cytotoxic agents in the extracellular matrix. Histological analysis of hematoxylin and eosin and masson's trichrome stained processed aortic samples shows complete decellularization with preservation of extracellular matrix microarchitecture at 120 h. Further, staining of tissue samples with DAPI demonstrates complete removal of DNA fragments. Quantitative evaluation of DNA in the decellularized aorta tissues demonstrated a significant (P < 0.01) decrease in DNA content as compared to native tissues. Collagen quantification assay indicate no significant (P> 0.05) difference in its content between native and decellularized caprine aorta. Tensile strength of the decellularized scaffolds decreased non-significantly (P > 0.05) when compared to native tissues. There was no significant (P > 0.05) difference in young's modulus of elasticity, stiffness and stretch ratio between native aortic tissues and decellularized aortic scaffolds. Histological and scanning electron microscopic examination of in vitro cultured scaffold demonstrated the cell viability and proliferation of primary chicken embryo fibroblasts. SPE treatment is thus capable of producing cytocompatible decellularized caprine aorta scaffold with preservation of extracellular matrix architecture for vascular tissue engineering and could be applied widely as one of the decellularization agent.

Isolation, Cryopreservation, and Characterization of iPSC-Derived Megakaryocytes.

Current research in the field of transfusion medicine is focused on developing innovative approaches to generate populations of functional megakaryocytes (MKs) ex vivo. This may open perspectives to establish alternative therapies for donor platelet transfusion in the management of thrombocytopenic patients and pave the way for novel regenerative approaches. Efficient cryopreservation techniques can provide the opportunity for long-term storage and accumulation of necessary amounts of MKs in a ready-to-use manner. However, in this case, besides the viability, it is crucial to consider the recovery of functional MK properties after the impact of freezing. In this chapter, the possibility to cryopreserve iPSC-derived MKs is described. In particular, the methods for a comprehensive analysis of phenotypic and functional features of MKs after cryopreservation are proposed. The use of cryopreserved in vitro-produced MKs may benefit to the field of transfusion medicine to overcome the lack of sufficient blood donors.

Pollen Grain Counting Using a Cell Counter.

The number of pollen grains is a critical part of the reproductive strategies in plants and varies greatly between and within species. In agriculture, pollen viability is important for crop breeding. It is a laborious work to count pollen tubes using a counting chamber under a microscope. Here, we present a method of counting the number of pollen grains using a cell counter. In this method, the counting step is shortened to 3 min per flower, which, in our setting, is more than five times faster than the counting chamber method. This technique is applicable to species with a lower and higher number of pollen grains, as it can count particles in a wide range, from 0 to 20,000 particles, in one measurement. The cell counter also estimates the size of the particles together with the number. Because aborted pollen shows abnormal membrane characteristics and/or a distorted or smaller shape, a cell counter can quantify the number of normal and aborted pollen separately. We explain how to count the number of pollen grains and measure pollen size in Arabidopsis thaliana, Arabidopsis kamchatica, and wheat (Triticum aestivum).

Human Liver Microsomes Immobilized on Magnetizable Beads: A Novel Approach to Study In Vitro Drug Metabolism.

Human liver microsomes (HLM) are a commonly used tool to study drug metabolism in vitro. Typical experiments conducted using suspensions of HLM can be challenging to separate from the incubation solution without lengthy ultracentrifugation steps. Magnetizable beads coated with silica (MGBS) were found to bind strongly to HLM, which could then be isolated and purified using a magnet. Binding of HLM to the MGBS (HLM-MGBS) was demonstrated to be mediated by strong interactions between microsomal phospholipids and MGBS, as artificially prepared phosphatidylcholine (PC) liposomes could be more efficiently captured by the MGBS. HLM-MGBS complexes retained functional cytochrome P450 and uridine-diphosphate-glucuronosyltransferase (UGT) activity as indicated by CYP2C8-mediated amodiaquine de-ethylation, CYP3A4-mediated midazolam 1'hydroxylation, UGT1A1-mediated glucuronidation of estradiol, UGT1A9-mediated glucuronidation of propofol, and UGT2B7-mediated glucuronidation of zidovudine. When comparing suspension HLM alone with HLM-MGBS complexes containing equivalent amounts of HLM, the intrinsic clearance (CLint) of CYP450 substrates was comparable; however, CLint of UGT1A1, UGT1A9, and UGT2B7 was increased in the HLM-MGBS system between 1.5- and 6-fold. HLM-MGBS used in an incubation could also be readily replaced with fresh HLM-MGBS to maintain the presence of active enzymes. Thus, HLM-MGBS demonstrate increased in vitro metabolic efficiency and manipulability, providing a new platform for determination of accurate metabolic parameters. SIGNIFICANCE STATEMENT: The following work describes the strong binding of HLM to magnetizable beads. In addition, the preservation of enzyme activity on the bound HLM provides a novel means to conduct preclinical metabolism studies.

High-throughput single-cell live imaging of photobiomodulation with multispectral near-infrared lasers in cultured T cells.

SIGNIFICANCE: Photobiomodulation is a well-established therapeutic modality. However, the mechanism of action is poorly understood, due to lack of research in the causal relationship between the near-infrared (NIR) light irradiation and its specific biological effects, hindering broader applications of this technology. AIM: Since biological chromophores typically show several absorption peaks, we determined whether specific effects of photobiomodulation are induced with a combination of two wavelengths at a certain range of irradiance only, rather than a single wavelength of NIR light. APPROACH: In order to analyze a wide array of combinations of multispectral NIR light at various irradiances efficiently, we developed a new optical platform equipped with two distinct wavelengths of NIR lasers by high-throughput multiple dosing for single-cell live imaging. Two wavelengths of 1064 and 1270 nm were selected based on their photobiomodulatory effects reported in the literature. RESULTS: A specific combination of wavelengths at low irradiances (250 to 400 mW / cm2 for 1064 nm and 55 to 65 mW / cm2 for 1270 nm) modulates mitochondrial retrograde signaling, including intracellular calcium and reactive oxygen species in T cells. The time-dependent density functional theory computation of binding of nitric oxide (NO) to cytochrome c oxidase indicates that the illumination with NIR light could result in the NO release, which might be involved in these changes. CONCLUSIONS: This optical platform is a powerful tool to study causal relationship between a specific parameter of NIR light and its biological effects. Such a platform is useful for a further mechanistic study on not only photobiomodulation but also other modalities in photomedicine.

Antibody-modified magnetic nanoparticles as specific high-efficient cell-separation agents.

Separation of tumor cells is a promising approach that helps not only in early detection of cancer but also as an efficient tool that holds great importance in prohibiting cancer cell mutation, drug resistance to treatments, and in granting successful adjuvant therapies. As one of the highly efficient processes for the separation of single cells, tumor cells, and specific proteins from fresh whole blood, a magnetic iron oxide nanoparticle (IONP)-based immunomagnetic separation technique has been developed in this article. The synthesized IONPs were modified with antibodies (Abs) against human epithelial growth factor receptor 2 (HER2), which is overexpressed and/or amplified in about 15% of breast cancer patients with several types of human cancer cells. The prepared Ab-conjugated IONPs (Ab-IONPs) attach HER2-positive cancer cells exclusively and can serve as specific high-efficient single-cell separation agents. The results showed that the magnetic IONPs have been successfully attached to the Abs via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide linkers. Maximum targeting efficiency of the Ab-IONP complex, which was 94.5 ± 0.8% for BT474 and 70.6 ± 0.4% for mixture of cells (BT474 and MCF7), was achieved with a minimum amount of Abs, to provide an economically efficient single-cell detection device.

Necrostatin-1 supplementation enhances young porcine islet maturation and in vitro function.

BACKGROUND: Necroptosis has been demonstrated to be a primary mechanism of islet cell death. This study evaluated whether the supplementation of necrostatin-1 (Nec-1), a potent inhibitor of necroptosis, to islet culture media could improve the recovery, maturation, and function of pre-weaned porcine islets (PPIs). METHODS: PPIs were isolated from pre-weaned Yorkshire piglets (8-15 days old) and either cultured in control islet culture media (n = 6) or supplemented with Nec-1 (100 µM, n = 5). On days 3 and 7 of culture, islets were assessed for recovery, insulin content, viability, cellular composition, GLUT2 expression in beta cells, differentiation of pancreatic endocrine progenitor cells, function, and oxygen consumption rate. RESULTS: Nec-1 supplementation induced a 2-fold increase in the insulin content of PPIs on day 7 of culture. When compared to untreated islets, Nec-1 treatment doubled the beta- and alpha-cell composition and accelerated the development of delta cells. Additionally, beta cells of Nec-1-treated islets had a significant upregulation in GLUT2 expression. The enhanced development of major endocrine cells and GLUT2 expression after Nec-1 treatment subsequently led to a significant increase in the amount of insulin secreted in response to in vitro glucose challenge. Islet recovery, viability, and oxygen consumption rate were unaffected by Nec-1. CONCLUSION: This study underlines the importance of necroptosis in islet cell death after isolation and demonstrates the novel effects of Nec-1 to increase islet insulin content, enhance pancreatic endocrine cell development, facilitate GLUT2 upregulation in beta cells, and augment insulin secretion. Nec-1 supplementation to culture media significantly improves islet quality prior to xenotransplantation.

Enzymatic Isolation, Amplification and Characterization of Dental Pulp Stem Cells.

The dental pulp represents an easily accessible source of adult dental pulp stem cells (DPSCs). The preferred approach to DPSC isolation is enzymatic digestion. However, the duration of the enzymatic activity is crucial. The purpose of this study was to isolate the DPSC populations using this method, characterize their biological properties and proliferation capacity, and to determine their ability to differentiate into mature cells. Before enzymatic digestion using 0.05% trypsin, we used the homogenization method in order to obtain a fine homogenate from the solid pulp tissue. The stem cells were cultivated in modified cultivation medium for mesenchymal adult progenitor cells containing 2% foetal bovine serum, growth factors and insulin-transferrin-selenium supplement. We were successfully able to isolate 10 populations of DPSCs. The vitality of DPSCs did not drop below 90 %. However, the DPSCs showed a significant decrease in the relative telomere length number with increasing passaging (P < 0.05). Isolated DPSCs highly expressed the CD markers: CD29, CD44, CD90, CD13, CD73 and CD166. In contrast, CD markers CD31, CD106, CD34 and CD45 were negative or low positive. We confirmed the high osteogenic and chondrogenic potential of the isolated stem cells. Isolated DPSCs did not show signs of cell degeneration or spontaneous differentiation during the entire cultivation. In addition, we were able to shorten the enzyme activity duration, and we were the first to demonstrate trypsin as the enzyme used for the enzymatic digestion method with the viability over 90 % of isolated DPSCs using this method.

Chromium 10× Single-Cell 3' mRNA Sequencing of Tumor-Infiltrating Lymphocytes.

Chromium 10× 3' V2 protocol is a 3' end counting single-cell mRNA sequencing protocol that allows to process and sequence RNA from thousands of cells in parallel. Chromium10× by 10× Genomics is an emulsion-based device that enables to compartmentalize single cells along with sets of uniquely barcoded primers and reverse transcription reagents into nanoscale droplets that are used as reaction chambers to generate barcoded full-length cDNA from single cells. After RT reaction single-stranded barcoded cDNAs are pooled together and processed to generate sequencing libraries compatible with the standard Illumina platforms. Here we show in detail the main steps of the protocol applied to the analysis of tumor-infiltrating T lymphocytes (TILs). The main steps are cell preparation, cDNA synthesis, library construction, and sequencing.This protocol refers specifically to the CG00052_SingleCell3_ReagentKitv2UserGuide_RevD downloadable from 10× Genomics website ( https://www.10xgenomics.com ) and does not substitute it. Always refer to this guide, paying attention to updates and revisions.

The Neurosphere Assay (NSA) Applied to Neural Stem Cells (NSCs) and Cancer Stem Cells (CSCs).

The discovery of neural stem cells (NSCs) in the mammalian brain has raised many expectations as these unique cells might recapitulate different neurological diseases, including brain tumors, both from a functional and molecular perspective. Proper in vitro culturing of NSCs has emerged as a critical methodological issue, given that it should preserve the in vivo features of NSCs, with particular emphasis on cell heterogeneity. At the same time, the methodology for NSC culturing should allow the production of large amounts of cells to be exploited not only for prospective clinical applications but also for drug screening. Direct in vitro selection of NSCs and, very recently, cancer stem cells (CSCs) by means of defined serum-free conditions represents the most reliable methodology to obtain long-term expanding SC lines. Here we describe the methods currently employed to enrich for NSCs/CSCs based on the neurosphere assay (NSA) and their adaptation to specific assays for testing the efficacy of neuroactive compounds.

What are the effects of vitamin C on sperm functional properties during direct swim-up procedure?

SummaryDirect swim-up procedure is widely used to separate the motile competent spermatozoa from the antioxidant-rich semen. Subsequently, spermatozoa become more vulnerable to reactive oxygen species (ROS) due to their cytological characteristics. The effect of vitamin C, a highly concentrated antioxidant in the semen, on direct swim-up-enriched sperm population is not fully investigated. Therefore, the aim of the present study was to assess the effect of vitamin C on sperm functional properties during direct swim-up procedure. Semen samples were collected from 22 participants. Each semen sample was divided into several aliquots. The first portion was overlaid with sperm medium without ascorbic acid (0 µM AA). The second and third fractions were overlaid with sperm medium supplemented with 300 µM and 600 µM AA; respectively. After 1 h of incubation, basic sperm parameters, intracellular ROS levels, acrosome reaction, chromatin integrity, and glucose uptake were assessed. Swim-up without AA significantly increased the percentage of ROS(+) spermatozoa compared with the raw semen (P<0.01). Interestingly, swim-up with 300 µM AA did not increase the percentage of ROS(+) sperm compared with the raw semen. In parallel, the percentage of sperm with altered chromatin integrity was significantly lower in the 300 µM AA group compared with that in the raw semen (P<0.05). These findings suggest that supplementation of vitamin C to sperm medium could be beneficial for direct swim-up-derived spermatozoa.

Transcriptomics of Arabidopsis sperm cells at single-cell resolution.

KEY MESSAGE: We present a detailed protocol for isolation of single sperm cells and transcriptome analysis to study variation in gene expression between sperm cells. Male gametophyte development in flowering plants begins with a microspore mother cell, which upon two consecutive cell divisions forms a mature pollen grain containing a vegetative nucleus and two sperm cells. Pollen development is a highly dynamic process, involving changes at both the transcriptome and epigenome levels of vegetative nuclei and the pair of sperm cells that have their own cytoplasm and nucleus. While the overall transcriptome of Arabidopsis pollen development is well documented, studies at single-cell level, in particular of sperm cells, are still lacking. Such studies would be essential to understand whether and how the two sperm cells are transcriptionally different, in particular once the pollen tube grows through the transmitting tissue of the pistil. Here we describe a detailed protocol for isolation of single sperm cells from growing pollen tubes and analysis of their transcriptome.

Development of Bispecific Antibody Derivatives for Cancer Immunotherapy.

Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which redirects and potently activates T cell immune responses. Recently, we described an additional novel bispecific antibody derivative, termed RTX-CD47, which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here, we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.

Using Acutely Dissociated and Purified Oligodendrocyte Precursor Cells for High-Throughput Drug Screening to Identify Compounds that Promote Oligodendrocyte Differentiation.

Multiple sclerosis (MS) is an autoimmune disease that involves an immune-mediated inflammatory response in the central nervous system and optic nerve resulting in demyelination and neural degeneration, the cause of which is unknown. The adult central nervous system has the capacity to remyelinate axons by generating new oligodendrocytes (OLs). To identify clinical candidate compounds that may promote remyelination, we have developed a high-throughput screening (HTS) assay to identify compounds that promote the differentiation of oligodendrocyte precursor cells (OPCs) into OLs. Using acutely dissociated and purified rat OPCs coupled with immunofluorescent image quantification, we have developed an OL differentiation assay. Building on OPC culturing techniques developed over the past 30 years, we have scaled up the isolation and purification process to generate sufficient quantities for HTS. We then describe the use of these acutely derived OPCs in an assay designed to identify compounds that promote differentiation into OLs. We have validated this assay with a known promoter of differentiation, thyroid hormone, and subsequently used the assay to screen the NIH clinical collection library (Lariosa-Willingham, et al., 2016). © 2018 by John Wiley & Sons, Inc.

Detecting Autologous Blood Transfusion in Doping Control: Biomarkers of Blood Aging and Storage Measured by Flow Cytofluorimetry.

BACKGROUND: Blood transfusions are banned by the World Anti-Doping Agency as a form of "blood doping". A method of detection of homologous blood transfusion (HBT) has been implemented by the accredited anti-doping laboratories worldwide; however, no internationally recognized method has been finalized so far for the direct detection of autologous blood transfusions, which can at present be revealed only by targeted longitudinal profiling of key blood parameters. METHODS: The present article reports the results of an investigation aimed to pre-select potential biomarkers of blood aging and storage that can be measured to identify the presence in the sample of reinfused blood. Microparticles from platelets and erythrocytes, erythrocytes size and density, annexin V (as a marker of phosphatidylserine externalization), and the membrane surface antigens CD 55 and CD 59, were specifically considered as potential biomarkers and measured by flow cytofluorimetric techniques. RESULTS AND CONCLUSION: Our results indicate that the parameters more strongly affected by the ex vivo storage of whole blood are erythrocytes size and density, annexin V and microparticles. Although the real diagnostic value of the proposed biomarkers shall obviously be confirmed by further studies carried out on blood samples collected after an actual autologous blood transfusion, these results appear very encouraging towards the development of a direct method for detecting autologous blood transfusion in sport doping.

Isolation of HL-60 cancer cells from the human serum sample using MnO2-PEI/Ni/Au/aptamer as a novel nanomotor and electrochemical determination of thereof by aptamer/gold nanoparticles-poly(3,4-ethylene dioxythiophene) modified GC electrode.

Herein, aptamer-modified self-propelled nanomotors were used for transportation of human promyelocytic leukemia cells (HL-60) from a human serum sample. For this purpose, the fabricated manganese oxide nanosheets-polyethyleneimine decorated with nickel/gold nanoparticles (MnO2-PEI/Ni/Au) as nanomotors were added to a vial containing thiolated aptamer KH1C12 solution as a capture aptamer to attach to the gold nanoparticles on the surface of nanomotors covalently. The aptamer-modified self-propelled nanomotors (aptamerKH1C12/nanomotors) were then separated by placing the vial in a magnetic stand. The aptamer-modified self-propelled nanomotors were rinsed three times with water to remove the non-attached aptamers. Then, the resulting aptamerKH1C12/nanomotors were applied for the on-the-fly" transporting of HL-60 cancer cell from a human serum sample. To release of the captured HL-60 cancer cells, the complementary nucleotide sequences of KH1C12 aptamer solution (releasing aptamer) that has a with capture aptamer was added to phosphate buffer solution (1 M, pH 7.4) containing HL-60/aptamerKH1C12/nanomotors. Because of the high affinity of capture aptamer to complementary nucleotide sequences of aptamerKH1C12, the HL-60 cancer cells released on the surface of aptamerKH1C12/nanomotors into the solution. The second goal of the present work was determining the concentration of HL-60 cancer cell in the human serum samples. The electrochemical impedance spectroscopy technique (EIS) was used for the determination of HL-60 cancer cell. The concentration of separated cancer cell was determined by aptamer/gold nanoparticles-poly(3,4-ethylene dioxythiophene) modified GC electrode (GC/PEDOT-Aunano/aptamer KH1C12). The proposed aptasensor exhibited a good response to the concentration of HL-60 cancer cells in the range of 2.5 × 101 to 5 × 105 cells mL-1 with a low limit of detection of 250 cells mL-1.

Continuous and Quantitative Purification of T-Cell Subsets for Cell Therapy Manufacturing Using Magnetic Ratcheting Cytometry.

T-cell-based immunotherapies represent a growing medical paradigm that has the potential to revolutionize contemporary cancer treatments. However, manufacturing bottlenecks related to the enrichment of therapeutically optimal T-cell subpopulations from leukopak samples impede scale-up and scale-out efforts. This is mainly attributed to the challenges that current cell purification platforms face in balancing the quantitative sorting capacity needed to isolate specific T-cell subsets with the scalability to meet manufacturing throughputs. In this work, we report a continuous-flow, quantitative cell enrichment platform based on a technique known as ratcheting cytometry that can perform complex, multicomponent purification targeting various subpopulations of magnetically labeled T cells directly from apheresis or peripheral blood mononuclear cell (PBMC) samples. The integrated ratcheting cytometry instrument and cartridge demonstrated enrichment of T cells directly from concentrated apheresis samples with a 97% purity and an 85% recovery of magnetically tagged cells. Magnetic sorting of different T-cell subpopulations was also accomplished on chip by multiplexing cell surface targets onto particles with differing magnetic strengths. We believe that ratcheting cytometry's quantitative capacity and throughput scalability represents an excellent technology candidate to alleviate cell therapy manufacturing bottlenecks.