Single B Cell Sorting Protocol and FAQs

Single B Cell Sorting is a high-precision experimental technique, which is usually used to analyze and isolate individual B cells for in-depth analysis of genes, proteins or functions. B cells are an important cell type in the immune system, which are mainly responsible for producing antibodies and participating in the humoral immune response. Due to the diversity of B cells and their complex roles in immune responses, studies at the single cell level can reveal the specific characteristics and functions of each B cell.

The Principle of Single B Cell Sorting

Flow Cytometry Sorting (FACS)

B cell surface markers (such as CD19, CD20, IgD / IgM, etc.) and target antigen binding ability (such as fluorescently labeled antigen probes) were detected by laser excitation of fluorescently labeled antibodies. Forward scattering (FSC) and lateral scattering (SSC) were used to distinguish cell size and particle size, and the target B cells were screened by fluorescence signal.

Droplet Formation Technology

The liquid flow is broken into droplets containing a single cell by high-frequency vibration, and the charged droplets are sorted by electric field deflection.

Microfluidic Sorting

In the chip, cells are controlled by fluid mechanics or dielectric power, and single B cells are captured by image recognition technology.

The Steps of Single B Cell Sorting

Sample Preparation

Cell source

Cell samples come from a variety of sources, including cells extracted from tissues (such as tumor tissues, liver, kidney, etc.), blood, and cell cultures. Cells from different sources may have different sorting requirements and processing methods.

Cell Digestion

When the cell source is a tissue sample, the tissue needs to be digested into a single cell suspension by enzymatic hydrolysis. Common digestive enzymes include trypsin, collagenase and DNase. During the digestion process, the concentration and reaction time of the enzyme need to be accurately controlled to ensure that the cells are not excessively damaged.

Filtration

In order to remove cell masses and debris that may be produced during tissue digestion, a 40μm filter is often used for filtration. This ensures that the sorted cells are a single cell suspension, avoiding interference with subsequent experiments.

Cell Counting And Activity Detection

Before sorting, cells need to be counted and their activity detected. The commonly used method is trypan blue staining, which can clearly distinguish dead cells from live cells and ensure that the proportion of live cells remains above 85 %.

Cell Staining (Optional)

Fluorescent labeling

In flow cytometry sorting (FACS), in order to identify specific cell populations, fluorescently labeled antibodies are usually used to label specific antigens on the cell surface. Titration experiments were carried out to determine the optimal concentration for incubating each fluorescent antibody. By fluorescent labeling, target cells and non-target cells can be distinguished.

Fluorescein selection

It is very important to select the appropriate fluorescein. Common fluorescent dyes include FITC (green), PE (orange), APC (red), etc.When selecting fluorescent dyes, it is necessary to ensure that the emission spectra of each fluorescein do not overlap.

Incubation and washing

After staining, it is usually incubated to ensure that the fluorescent antibody is fully combined with the cell surface antigen. After incubation, it is necessary to use PBS or a special flow buffer for washing to remove unbound fluorescent dyes to prevent them from interfering with the sorting results.

Cell Sorting

Flow cytometry sorting (FACS)

Principle: Flow cytometry sorting technology is based on the characteristics of cell size, morphology and fluorescent labeling to sort cells. Flow cytometry (e.g. BD FACSAria) was used to distinguish cell populations by laser irradiation of samples and by scattered light (FSC / SSC). Dead cells, cell clumps and debris are usually excluded. Single positive and FMO (Fluorescence Minus One) control were used to adjust the spectral overlap and control the compensation.

Process: Set different gating strategies, such as FSC and SSC as the standard for coarse screening, and then further accurately select the target cell subsets through fluorescence signals. Single cells can be collected directly into PCR tubes, 96-well plates or culture media.

Magnetic bead sorting (MACS)

Principle : Magnetic bead sorting technology uses magnetic separation column for sorting after labeling molecules on the surface of target cells (such as CD3 + T cells) with specific antibodies. By capturing the cells labeled with magnetic beads under the action of a magnetic field, positive or negative selection is performed.

Characteristics : Magnetic bead sorting method is suitable for large-scale cell sorting, and the processing speed is faster, but the purity is usually lower than that of flow cytometry sorting, which is suitable for applications that do not require extremely high purity.

Microfluidic single cell sorting

Principle : Microfluidic chips (such as 10X Genomics) use tiny fluid channels to precisely manipulate cells. Through water-in-oil droplet technology, each droplet contains a single cell, ensuring that each cell is given a separate environment, and single cell sorting can be performed and a library can be quickly constructed.

Advantages : It is suitable for large-scale, high-throughput single-cell analysis, can effectively reduce operating errors, and is suitable for single-cell RNA sequencing and other research.

Quality Control and Storage

Microscopic examination

The sorted cells need to be examined by microscope to confirm the morphology and integrity of the cells. Usually check whether the cells have obvious morphological damage or debris.

Single cell activity detection

In order to ensure the effectiveness of subsequent experiments, the activity status of the selected single cells is usually detected. The commonly used method is to determine the quality of RNA (e.g., RNA integrity value, RIN) to ensure the integrity of RNA.

Storage mode

The sorted single cells can choose different storage modes according to their needs. Short-term storage can be placed in a refrigerator at 4℃, while long-term storage requires the addition of cryopreservation solution and placed in -80℃ or liquid nitrogen to prevent cell death or RNA degradation.

Follow-up Analysis

Single-cell sequencing

Single cells that have been sorted and stored usually undergo further molecular biological analysis, the most common of which is single-cell RNA sequencing (scRNA-seq). In addition, there are applications such as single-cell ATAC-seq (analysis of chromatin openness) and single-cell proteomics.

Single cell culture

The sorted single cells can also be used for clone culture to study the behavior of specific cell populations such as stem cells and cancer stem cells. It can also be used for cell model establishment and drug screening.

Functional experiments

The cells after single cell sorting can also be used for functional experiments such as immune detection, drug screening, phenotypic analysis, etc., to study the biological characteristics or drug response of cells.

Figure 1. The workflow of research：A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations. (Reference source: A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations.)

Alpha Lifetech provides single B cell sorting assay to provide customers with efficient and accurate single cell analysis services. Through advanced flow cytometry technology, we can accurately isolate and screen target B cells to help customers obtain high-quality experimental data in the fields of immunological research, vaccine development and antibody screening.

FAQ

1. The low efficiency of cell sorting leads to the insufficient number of target cells.

The cell concentration of the sample before sorting was increased. Check whether the nozzle is blocked. Adjust the sorting parameters of the flow cytometer, such as flow rate and sorting mode. Optimize the use of antibodies to ensure that target B cells can be effectively labeled. Flow sorting is assisted by other technologies (such as magnetic bead sorting).
2. The sorted B cells may lose their function or lower activity.

Solutions : Ensure that the sorting process is as gentle as possible to reduce the loss of cell function. Adding 2 mM EDTA and 1 % BSA to the sorting buffer can increase cell activity. Appropriate culture conditions and medium were used to maintain cell activity and function. In the short time after sorting, cells are used for downstream experiments to avoid functional loss.
3. If the selected B cell surface markers are not accurate, it may lead to unsatisfactory sorting results.

Specific fluorescently labeled antibodies are used to stain B cell surface marker molecules. Commonly used antibodies include antibodies against B cell-specific markers such as CD19, CD20, and CD38. Check the specificity and reactivity of the antibody to avoid cross-reaction. Using multiple labeling strategies, combined with multiple surface markers to improve the accuracy of sorting.
4. Impurities (such as bubbles, stray particles) may appear in the process of flow cytometry sorting, interfering with cell sorting.

Ensure that the optical path of the flow cytometer is clean and avoid stray light interference. Regularly calibrate the equipment to ensure the accuracy of the flow cytometer.
5. The low efficiency of cell sorting leads to the insufficient number of target cells.

The cell concentration of the sample before sorting was increased. Check whether the nozzle is blocked. Adjust the sorting parameters of the flow cytometer, such as flow rate and sorting mode. Optimize the use of antibodies to ensure that target B cells can be effectively labeled. Flow sorting is assisted by other technologies (such as magnetic bead sorting).

reference

[1] Lei L, Tran K, Wang Y, et al. Antigen-Specific Single B Cell Sorting and Monoclonal Antibody Cloning in Guinea Pigs. Front Microbiol. 2019;10:672. Published 2019 Apr 23. doi:10.3389/fmicb.2019.00672

[2] Tomlinson JE, Wagner B, Felippe MJB, Van de Walle GR. Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood. Vet Immunol Immunopathol. 2018;199:22-31. doi:10.1016/j.vetimm.2018.03.010

[3] Lin W, Liang WC, Nguy T, et al. Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology. PLoS One. 2020;15(12):e0244158. Published 2020 Dec 21. doi:10.1371/journal.pone.0244158

[4] Zhou Y, Liu Z, Wang Z, et al. Single-Cell Sorting of HBsAg-Binding Memory B Cells from Human Peripheral Blood Mononuclear Cells and Antibody Cloning. STAR Protoc. 2020;1(3):100129. Published 2020 Oct 16. doi:10.1016/j.xpro.2020.100129

[5] Nogales-Gadea G, Saxena A, Hoffmann C, et al. Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells. J Vis Exp. 2015;(100):e52830. Published 2015 Jun 5. doi:10.3791/52830

[6] Ibrahim SF, van den Engh G. Flow cytometry and cell sorting. Adv Biochem Eng Biotechnol. 2007;106:19-39. doi:10.1007/10_2007_073