Preservation methods and precautions for common flow-through samples

Flow cytometry plays an important role in cell counting, cell antigen detection, cell function detection, cell nuclear acidic property analysis and other experiments. Compared with other detection methods, it has the advantages of precise target, quantitative index, sample saving, high throughput and other detection advantages.

As a crucial part of flow cytometry, sample preparation and preservation play a pivotal role in the success or failure of the experiment. Below is a summary of common sample preservation methods and precautions in flow cytometry.

First, there are several common samples for flow cytometry:

	Categorization	Hallmark
Fluid Sample	Bone marrow, peripheral blood, body fluids (pleural fluid, ascites, cerebrospinal fluid)	Cells are unicellular and require addition of anticoagulant and lysis of erythrocytes
Fluid Sample	In vitro culture of passaged cell line cells	Contains both adherent and suspension cells
Sample of entities	Non-immune system tissues: liver, heart, lungs, gastrointestinal tissue, brain tissue, etc.	Cells are tightly linked and require single-cell suspensions prepared by enzymatic digestion or grinding
Sample of entities	Immune system: thymus, spleen, lymph nodes, etc.	Cells are tightly linked and require single-cell suspensions prepared by enzymatic digestion or grinding

Each sample has its own characteristics, and the preservation and handling of common samples are summarized below:

Handling and preservation of fluid samples

name	Treatment	Method and duration of retention	caveat	appliance
Peripheral blood samples	Fresh samples were collected and mixed thoroughly with the anticoagulant, indicating the date and time of sample collection	Room temperature: dipotassium ethylenediaminetetraacetate can be stabilized for 12~24 h; glucose citrate solution can be stabilized for 72 h; sodium heparin anticoagulant can be stabilized for 48 h	If leukocyte counting, leukocyte sorting and flow cytometry tests share the same tube of sample, dipotassium ethylenediaminetetraacetate should be chosen as the anticoagulant	Immunophenotyping tests for hematologic disorders; white blood cell counts; leukocyte morphologic staining and classification
bone marrow sample	Human bone marrow samples: fresh samples collected and placed in sodium heparin anticoagulant	Normal samples for T-cell assay should be stored at room temperature with sodium heparin anticoagulation for 48 h; B-cell assay should be stored at 4℃ for 72 h; mononuclear and myeloid phenotypic assays can be stored at 4℃ for 48 h; and assays for highly differentiated lymphoma samples are recommended to be performed immediately after collection.	Other anticoagulants should not be used, as the ratio of bone marrow aspirate sample to glucose citrate solution is not optimal when it tends to lead to a change in pH, which reduces the activity of cells	Phenotypic analysis of immune system disorders; morphologic staining analysis
bone marrow sample	Mouse bone marrow sample: 1~2mL syringe blown into single cell suspension, lysed erythrocytes, resuspended in sterile RPMI culture medium and other tissue culture media	Live cells: single cell suspension can be stored for 24 h at 4℃ using staining buffer; fixed cells: fixed for 30 min at room temperature using 4% paraformaldehyde solution, washed by centrifugation, resuspension of cell precipitates in 1% formaldehyde phosphate buffer, stored for 1 week at 4℃.	Lysis of erythrocytes on ice, the length of erythrocyte lysis should not exceed 1 min; when the fixed cells are resuspended with 1% formaldehyde or paraformaldehyde buffer, the buffer should be used as it is prepared.	Cell sorting; in vitro culture and expansion; immunologic correlation studies; bone marrow immune cell phenotyping
Pleural fluid, ascites, cerebrospinal fluid samples	Standardized operations for sample collection, with sodium heparin, dipotassium ethylenediaminetetraacetic acid, and dextrose citrate solution as anticoagulants for pleural fluid and ascites samples; cerebrospinal fluid using culture media such as Transfix or transfer matrices	Thoracic/abdominal fluid samples can be stored at 4℃ for 24 h; cerebrospinal fluid can be stored at 4℃ for 24～48 h.	Selection of glucose citrate liquid anticoagulant overnight preservation of the sample cells are weaker and less active, pay attention to its and anticoagulant to maintain the appropriate ratio; cerebrospinal fluid is not applicable to any anticoagulant	Phenotypic analysis of immune system disorders; morphologic staining analysis
In vitro cultured cell lines	Suspension cells were collected directly; Adherent cells are collected after enzymatic digestion	Immediate detection, processed within 3 h at room temperature	Selection of appropriate buffers for different detection methods to avoid interference by some metal ions	Live cell AnnexinV apoptosis detection; cell death analysis; cell phenotype analysis, etc.
In vitro cultured cell lines	Suspension cells were collected directly; Adherent cells are collected after enzymatic digestion	Post-fixation storage: TUNEL flow-through fixative is fixed for 60 min at room temperature, washed by centrifugation, and then frozen with 90% FBS+10% DMSO, which can be stably stored for 2 months.	Cell fixation should be adequate; freezing cells with attention to gradient cooling	TUNEL flow-through assay; immobilization-resistant phenotypic antibody assay, etc.
In vitro cultured cell lines	Suspension cells were collected directly; Adherent cells are collected after enzymatic digestion	Post-fixation storage: 70% ice ethanol -20℃ fixed storage 1 week	Cell precipitation was resuspended using PBS, and pre-cooled anhydrous ethanol at -20℃ was added to a final concentration of 70%, and care was taken to mix the fixative as it was added to avoid clumping of cells.	Cell cycle assay; DNA probe assay, etc.

Handling and preservation of physical samples

name	Treatment	Method and duration of retention	caveat	appliance
Liver, heart, spleen, kidneys, brain tissue, lungs, stomach and intestines, and other less rigid tissues	Sieve milling treatment; enzymatic digestion treatment	Living tissue: tissue culture medium immersion, 4 ℃ can be stored for 24 ~ 48 h; placed in 0.9% sodium chloride solution, 4 ℃ environment can be stored for 24 h. Single-cell suspension: cell precipitation using HBSS buffer (containing calcium and magnesium ions) or whole medium resuspension at 4 ℃ can be stored for 24 h. Fixed cells: prepared into single-cell suspensions, the use of 4% paraformaldehyde solution fixed at room temperature for 30 min. Centrifugation and washing, 1% formaldehyde phosphate buffer resuspension of cell precipitation, 4 ℃ storage for 1 week	Grind the tissues with a sieve immersed in the medium, and use a softer latex rod or the piston end of a syringe to avoid physical damage to the cells; be careful not to over-digest the cells in enzymatic digestion; add HBSS buffer (containing calcium and magnesium ions) in time to terminate the digestion and centrifugal washing after enzymatic digestion, the buffer containing calcium and magnesium ions helps to maintain the viability of the cells.	Cell sorting; immune cell phenotyping; immune cell cytokine secretion function detection, etc.
Harder tissues such as lymph nodes, cartilage, and solid tumors	enzymatic digestion	Living tissue: tissue culture medium immersion, 4 ℃ can be stored for 24 ~ 48 h; placed in 0.9% sodium chloride solution, 4 ℃ environment can be stored for 24 h. Single-cell suspension: cell precipitation using HBSS buffer (containing calcium and magnesium ions) or whole medium resuspension at 4 ℃ can be stored for 24 h. Fixed cells: prepared into single-cell suspensions, the use of 4% paraformaldehyde solution fixed at room temperature for 30 min. Centrifugation and washing, 1% formaldehyde phosphate buffer resuspension of cell precipitation, 4 ℃ storage for 1 week	Grind the tissues with a sieve immersed in the medium, and use a softer latex rod or the piston end of a syringe to avoid physical damage to the cells; be careful not to over-digest the cells in enzymatic digestion; add HBSS buffer (containing calcium and magnesium ions) in time to terminate the digestion and centrifugal washing after enzymatic digestion, the buffer containing calcium and magnesium ions helps to maintain the viability of the cells.	Cell sorting; immune cell phenotyping; immune cell cytokine secretion function detection, etc.

In summary, different cells and tissues will have different treatment protocols and preservation methods depending on the content of the experiment, and optimal sample treatment protocols need to be developed according to specific problems and experimental needs.

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