Comparative Characteristic of the Methods of Isolation of Oocytes and Spermatozoa in Laboratory Animals (Review).

Over the past decade, there has been an increasing trend in the use of assisted reproductive technologies, which have significantly expanded the opportunities to overcome the problem of infertility. However, the problem of increasing the effectiveness of in vitro fertilization remains open. Isolation of germ cells from animals is a necessary process for various experimental studies. Animal germ cells can be used in experiments to study physical, chemical, genetic, immunological, and microbiological factors affecting reproduction efficiency and for the development of techniques that increase the effectiveness of in vitro fertilization. All of the above determines the relevance of studying existing methods of oocyte and sperm isolation for experimental in vitro studies. Here we discuss the existing methods of sperm and oocyte isolation from animals and their advantages and disadvantages, and also substantiate priority methods for use.

Protocol for isolating high-purity natural killer cells from peripheral blood of septic patients using magnetic cell separation.

In human sepsis, myelocytosis and concomitant lymphopenia complicate the study of peripheral blood natural killer (NK) cells. Here, we present a protocol for isolating NK cells from peripheral blood of septic patients using magnetic cell separation. We describe steps for the depletion of non-NK cells and NK cell enrichment. We then detail procedures for comparing the results from this protocol to results obtained through the isolation procedures using two commercially available kits for NK cell isolation. For complete details on the use and execution of this protocol, please refer to Coulibaly et al.1.

Protocol for optimized nasal mucosa sample processing to obtain high-quality scRNA-seq and scATAC-seq data.

Examining nasal mucosa samples is crucial for nasal cavity disease research and diagnosis. Simultaneously obtaining high-quality data for single-cell transcriptomics (single-cell RNA sequencing [scRNA-seq]) and epigenomics (single-cell assay for transposase-accessible chromatin using sequencing [scATAC-seq]) of nasal mucosa tissues is challenging. Here, we present a protocol for processing human nasal mucosa samples to obtain data for both scRNA-seq and scATAC-seq. We describe steps for extracting human nasal mucosa tissue, mechanical and enzymatic dissociation, lysis of red blood cells, and a viability assay. We then detail procedures for library preparation and quality control.

Protocol for the processing, cryopreservation, and biobanking of patient-derived cells and tissues.

Biobanking of patient-derived specimens offers unique opportunities for retrospective testing that could potentially contribute to diagnosing and evaluating clinical conditions, advancing personalized medicine and translational biomedical discovery. In this protocol, we detail the collection, processing, and cryopreservation of peripheral blood, bone marrow, and lymph nodes from patients with hematological malignancies. This protocol can be used for multiomics to gain cellular and molecular insights into blood cancers and to test the therapeutic potential of compounds for translational biomedical research. For complete details on the use and execution of this protocol, please refer to Lim et al.1 and Rijal et al.2.

A Novel Approach for Mucosal and Bulbar Olfactory Ensheathing Cells Isolation Based on the Non-adherent Subculture Technique.

Introduction: Olfactory ensheathing cells (OECs) are widely used in transplantation studies. The high purification of this unique cell type is valuable for medical applications. Although recent improvements in OECs isolation procedures opened a new era in this field, the high purification efficacy and viability rate are still of concern. The most widely used OECs isolation techniques can be broadly classified based on adherence properties, particularly in olfactory bulb-derived OEC isolation. Considering the invasive nature of harvesting OECs from human olfactory bulbs, a highly efficient purification of these cells from olfactory mucosa can benefit clinical trials. In this study, we isolated OECs from rats' olfactory bulbs and mucosa due to their differential adherence properties and compared them. Methods: Cell preparations were characterized by NGFR p75 and S100ß antibodies, the specific markers for OECs, using immunocytochemistry and western blot analysis, respectively. OECs morphology and viability were monitored over time by microscopy and MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide) assay. Results: We found that OECs could be purified from the olfactory mucosa using our suggested method as efficiently as the olfactory bulb. Both derived OECs showed high levels of NGFR p75 and S100ß expression, although the S100ß expression was higher in olfactory mucosa-derived OECs preparations (P<0.05). Moreover, there was no significant difference between the two sources in cell viability in our suggested protocol. Conclusion: Due to the non-invasive harvesting method, olfactory mucosa-derived OECs are preferred from a clinical point of view in transplantation studies.

Protocol for assessing murine cell doublet engagement and subsequent effects using flow cytometry and imaging flow cytometry.

Physical interactions between two immune cells or between immune and cancer cells play a major role in shaping the immune response in the tumor microenvironment, making them prime therapeutic targets for bispecific engagers. Here, we present a protocol for assessing murine cell doublet engagement and subsequent effects using flow cytometry and imaging flow cytometry. We describe steps for identifying bispecific cell engager antibodies at the cell-cell interface, doublet quantification, and characterizing cellular protein morphology and processes within the doublet. For complete details on the use and execution of this protocol, please refer to Shapir Itai et al.1.

Protocol for quantum dot-based cell counting and immunostaining of pulmonary arterial cells from patients with pulmonary arterial hypertension.

Currently, there is no protocol for growing and culturing primary pulmonary arterial cells (PACs) available from the Pulmonary Hypertension Breakthrough Initiative (PHBI). Here, we present a protocol for cultivating and maintaining three major PACs collected from patients with pulmonary arterial hypertension (PAH): endothelial (PAH-ECs), smooth muscle (PAH-SMCs), and adventitial cells (PAH-ADCs). We describe steps for obtaining PACs from PHBI, evaluating the growth of cells labeled with quantum dots (QDs), and staining endothelial cell (EC) markers for immunofluorescence imaging. For complete details on the use and execution of this protocol, please refer to Al-Hilal et al.1.

Protocol for lipid mediator profiling and phenotyping of human M1- and M2-monocyte-derived macrophages during host-pathogen interactions.

Here, we present a protocol for primary, human immune cell isolation and stimulation for lipid mediator profiling. We describe steps for the isolation of monocytes from human leukocyte concentrates via density centrifugation and differentiation/polarization toward M1- or M2-monocyte-derived macrophages (MDMs). We detail stimulation approaches of MDMs with live bacteria or influenza A virus for lipid mediator profiling and sample preparation for subsequent analysis, such as enzyme expression, mRNA analysis, or surface marker determination. For complete details on the use and execution of this protocol, please refer to Jordan et al.1.

Protocol for generating human CAR-engineered macrophages by Vpx-containing lentivirus.

Human-derived macrophages are notoriously difficult to infect with HIV-1-based lentiviruses, posing a limitation to the advancement of chimeric antigen receptor macrophage (CAR-M) therapy. Here, we present a protocol for generating human chimeric antigen receptor (CAR)-engineered macrophages using the viral protein Vpx (encoded by the Sooty Mangabey simian immunodeficiency virus [SIV] and HIV-2 lineages) incorporated into the lentivirus vector, which enhances infection efficiency. We describe steps for cell cultivation, lentivirus production, concentration, infection procedures, and efficiency assessments. This protocol provides a foundation to study macrophage manipulation, especially with CAR or other immune engagers. For complete details on the use and execution of this protocol, please refer to Gao et al.1.

Protocol to identify and isolate rare murine tumor-resident dendritic cell populations for low-input transcriptomic profiling.

Conventional type 1 dendritic cells (cDC1s) are critical for innate sensing of cancer, yet they are scarce in the tumor microenvironment (TME). Here, we present a protocol to identify and isolate cDC1 subsets from murine implantable tumors for subsequent transcriptomic profiling using a flow sorting-based strategy. We describe steps for cell culture of mouse tumors, tumoral growth, dissociation and isolation of tumoral cells, extracellular staining, and cell sorting. We then detail procedures for RNA isolation, mRNA library preparation, and sequencing. For complete details on the use and execution of this protocol, please refer to Papadas et al.1.