In this study, we optimized such an assay and successfully used it. Please see the attached protocol for standard platelet flow cytometry and a marker list that outlines platelet markers for both humans and mice. Wash the cells well and treat them with the selected activator, inhibitor, or. Cytometry protocol for directly staining mouse whole blood forflow cytometry next message.
Prepare cells in a manner that generates a single cell suspension. Peripheral blood mononuclear cell pbmc isolation and red cell lysis procedures introduction. Nielsen1 abstract absolute neutrophil count anc is used clinically to monitor physiological dysfunc. If you are unable to immediately read your samples on a cytometer, keep them shielded from light and in a refrigerator set at 48c. This protocol provides a basic guide for the isolation of peripheral blood mononuclear cells pbmc from whole blood and the isolation of splenocytes from spleen. Pbmcs isolated from whole blood through ficoll gradient centrifugation or rbc lysed whole blood or nonadherent cultured cells are readily available for flow cytometric analysis. Tenparameter immunophenotyping of human lysed whole blood with the attune nxt flow cytometer allows identification of multiple t cell subsets and myeloid cells flow cytometry multiplexing used to analyze metabolic activity and assess pharmaceutical compound toxicity as a highthroughput screening tool. Surface staining of mouse splenocytes and peripheral blood cells. Bd technical support protocol, 2002 use this method to detect cells bearing specific membrane. Preparation of peripheral blood protocol flow cytometry.
Incubate for 5 minutes at room temperature on a rotator. Given the importance of this unique t cell subset in. Begin by adding whole blood to fluorochromeconjugated monoclonal antibodies that bind specifically to cell surface antigens. A new simple whole blood flow cytometrybased method for. T regulatory cells tregs, formerly known as t suppressor cells, are a t cell subset with direct roles in both autoimmunity and responses to pathogens. Preparation of whole bone marrow for mass cytometry analysis. Tenparameter immunophenotyping of human lysed whole blood. Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold 4 o c.
The samples should be resuspended in cell staining buffer. Optimizing recovery of frozen human peripheral blood. Cd14 antigen, using a bd facs brand flow cytometer. Direct immunofluorescence staining of surface epitopes of cells and blood. Combine the recommended quantity of each primary antibody in an appropriate volume of flow cytometry staining buffer so that the final staining volume is 100 l i. Characterization of human monocyte subsets by whole blood. Note that while some lysis solutions are compatible with nowash staining, clearer populations are evident in our hands when a wash step is used. A protocol for the comprehensive flow cytometric analysis of.
In addition, we have outlined detailed protocols for carrying out both surface and intracellular staining in. Tenparameter immunophenotyping of human lysed whole. Top up 2ml microcentrifuge tube containing stain and blood with 1step fixlyse solution made to 1x in distilled water. Absolute counting of neutrophils in whole blood using flow. Do not exceed 5 minutes, as the white blood cells will begin to lyse beyond 5. Dilute blood in equal volumes of room temperature pbsbsa for example, add 3 ml of pbsbsa to 3 ml of blood. Tenparameter immunophenotyping of human lysed whole blood with the attune nxt flow cytometer allows identification of multiple t cell subsets and myeloid cells flow cytometry multiplexing used. Flow cytometry is now a widely used method for analyzing the. Tregs decrease inflammation via the secretion of immunosuppressive cytokines il10, tgfb and also through direct suppression of inflammatory effector t cells such as th1 and th17 cells. Seroblock have been developed which, when added to a staining protocol, can.
Nettenstrom l, alderson k, raschke ee, evans md, sondel pm, olek s, et al. I stained blood mice balbc cells for flow cytometry analysis. General notes assay specifications sample type singlecell suspensions, including human whole blood and peripheral blood mononuclear cells pbmc, mouse dissociated tissues, and cell lines. Perform fluorescence activated cell sorting facs, or flow cytometric analysis. Staining cell surface targets for flow cytometry thermo fisher. Direct immunofluorescence staining of whole blood using a lyse nowash procedure source. Leukocytes are the most commonly analyzed cells in flow cytometry. Freshly isolated adipocytes are a notoriously difficult cell type to study. Intracellular flow cytometry product list flow cytometry kits and cocktails bd pharmingen mouse th1th17 phenotyping kit ms icfcm ruo fitc, pe, percpcy5. Facs sample prep from whole brain tissue mouse flow. General notes assay specifications sample type singlecell suspensions, including human whole blood and peripheral blood mononuclear cells. Bd technical support protocol, 2002 use this method to detect cells bearing specific membrane antigens. Flow cytometry is used extensively to examine immune cells in nonlymphoid tissues.
Facs sample prep from whole brain tissue mouse mar 162009 im looking for a protocol for sample preparation for facs from whole brain. Flow cytometric analysis of normal and reactive spleen. Cells are usually stained in polystyrene round bottom 12 x 75 mm 2 falcon tubes. An optimized multiparameter flow cytometry protocol for human t regulatory cell analysis on fresh and viably frozen cells, correlation with epigenetic analysis, and comparison of cord and adult blood. An optimized multiparameter flow cytometry protocol for human t regulatory cell analysis on fresh and viably frozen cells. Proceed to step 8 for purified or biotinylated primary antibodies. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase pgkfviii lentiviral vectortransduced cells by directly visualizing fviii in cells after different gene. Carefully overlay whole blood onto an equal volume of separation media in a 15 ml conical. Optional block nonspecific fcmediated interactions. This protocol is provided by the immune tolerance network as is without. Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold 4 o c pbsbsa buffer.
Collect the blood samples from the participants into 3 ml ethylene diamine tetra acetic acid edta tubes. Detection of factor viii fviii in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. Surface and intracellular staining protocols for flow cytometry. Flow cytometry is a sensitive technique currently employed by pathologists for quantitative and qualitative evaluation of hematopoietic cells. To stain whole blood with antibodies to surface markers for flow cytometric analysis. This protocol is written such that whole blood is stained for flow cytometry before rbc lysis. Bd biosciences intracellular flow cytometry product list. General procedure for flow cytometry using a conjugated primary antibody. Cd11b antigen, using a bd facs brand flow cytometer. Monoclonal mouse antihuman reagent for identification of cells. Absolute counting of neutrophils in whole blood using flow cytometry marion e. Immunophenotyping of whole peripheral blood wpb by flow cytometry. At the end of the cell treatment, immediately mix one volume of the warmed fix buffer with one volume of the suspended pbmcs.
Platelet staining in mouse blood for facs analysis. Bd phosflow protocol for human pbmcs bd biosciencesus. Direct immunofluorescence staining of whole blood using a. Monoclonal mouse antihuman reagent for identification of. We developed a protocol for the flow cytometric analysis of nonlymphoid tissues, including methods of tissue preparation, a 10fluorochrome panel for cell staining, and a standardized gating. Allowing for this difference in anc definition, the flow cytometry counting assays using ly6g can be used reliably in the research laboratory to quantify mouse anc from a small volume of blood. Cytometry protocol for directly staining mouse whole blood for flow cytometry iwei feng ifeng at mon sep 10 20. Baseline jak phosphorylation profile of peripheral blood. Surface staining of mouse splenocytes and peripheral blood. Protocol for flow cytometry on mononuclear cells from mouse. Harvest, wash the cells and adjust cell suspension to a concentration of 15 x 10 6 cellsml in ice cold pbs, 10% fcs, 1% sodium azide. Next, treat the stained sample with facs lysing solution to lyse erythrocytes under gentle hypotonic conditions while preserving the leucocytes. Discover more with our introduction to flow cytometry.
This protocol of fresh whole bm preparation may be used for 1, cytof analysis to discover unidentified cell populations from whole bm, 2, investigating whole bm defects for patients. Add 100 l of whole blood that was collected in a heparinized or edta coated blood collection tube. Apr 23, 2004 flow cytometry is a sensitive technique currently employed by pathologists for quantitative and qualitative evaluation of hematopoietic cells. How to differentiate tregulatory cells tregs by flow cytometry. Defining anc based on ly6g expression produces a 15% higher estimate than the dual protocol. Preparation of cells for flow cytometry for further information. Alternatively, whole blood can be lysed in bulk before staining using a ratio of 2 ml. Protocol for flow cytometry on mononuclear cells from mouse blood mar262014 hallo all. Adherent cultured cells or cells present in the solid organs should be first made into a single cell suspension before flow analysis by using enzymatic digestion or.
Thp1 cells stained with mouse antihuman cd11a blue or mouse igg2a. The cells will not lyse correctly if the solution is cold. Wash the cells well and treat them with the selected activator, inhibitor, or combination. Jun 19, 2019 this protocol of fresh whole bm preparation may be used for 1, cytof analysis to discover unidentified cell populations from whole bm, 2, investigating whole bm defects for patients with blood disorders such as leukemia, 3, assisting optimization of fluorescenceactivated flow cytometry protocols that utilize fresh whole bm. Mouse immunophenotyping and flow cytometry protocol version date 4272012 materials. Prepare cells as described in cell preparation protocols for flow cytometry found in our best protocols section. Strict adherence to time and temperature recommendations for fixation, permeabilization, and staining is necessary for optimal. Developing a flow cytometry protocol and workflow will take some troubleshooting as there are. Bd phosflow protocol for human whole blood samples cytobank. Cytometry cas hood for ariaiii cell sorter and uv lights. Whole blood samples may be used undiluted unless the cell count is high, e. A new simple whole blood flow cytometrybased method for simultaneous identification of activated cells and quantitative evaluation of cytokines released during activation.
Here, we describe a method to rapidly identify cd3. Centrifuge at 300400g for 30 minutes in a 20 o c temperature controlled centrifuge with no brake. Consensus protocol for the flow cytometric immunophenotyping of. Introduction to flow cytometry flow cytometry is a popular laserbased technology. Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Strict adherence to time and temperature recommendations for fixation, permeabilization, and staining is necessary for optimal resolution of. Staining procedure for all directly conjugated reagents whole. Harvest, wash the cells and adjust cell suspension to a concentration of 15 x 10 6 cellsml in ice cold pbs. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. Isolate peripheral blood mononuclear cells pbmcs by density gradient separation eg, using a cpt tube from whole blood. Preparation of human peripheral blood mononuclear cells pbmc leukocytes are the most commonly analyzed cells in flow cytometry. Using a phosphospecific whole blood flow cytometric method, we measured the baseline phosphorylation levels of all known jaks i. A good correlation was demonstrated between flow cytometry. Collect blood 75 microliters into 1ml pbs containing 5 microm edta 10 microliters of 0.
Flow cytometry is now a widely used method for analyzing the expression of cell surface and intracellular. The removal of red blood cells and fixation of white blood cells with lysis solution is an important step in this protocol as the presence of red blood cells can interfere with flow cytometry 21, 22. See appendix 5 for a complete list of validated cell types. Carefully overlay whole blood onto an equal volume of separation media in a 15 ml conical centrifuge tube. Blood human, mouse or rat fcm lysing solufor each 1 ml of blood, add 14 ml of room temperature tion sc3621 to lyse the red blood cells. Resuspend stained cells in an appropriate volume of flow cytometry staining buffer.
As human blood is potentially infectious, the sample setup should be performed in a biohazard hood. Peripheral blood mononuclear cell pbmc isolation and red. Introduction measurement of proten ei xpressoi n via flow cytometry offers. Current protocols in cytometry wiley online library. Flowcytometry platform for intracellular detection of fviii. Place 1 x 105 106 in a 12 x 75 mm tube or a 96well round bottom plate. Direct immunofluorescence staining of whole blood using a lyse wash procedure source. Next, treat the stained sample with facs lysing solution to lyse erythrocytes. The recovered preparations contain myeloid cells, lymphocytes, erythrocytes, and platelets.
Immunophenotyping of whole peripheral blood wpb by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. However, there is a current need for a simple, single. Protocol cell surface flow cytometry staining of whole blood. Leukocytes can be obtained from whole blood and a variety of tissues, such as spleen, lymph node, bone marrow and thymus. Here, we will discuss the various staining buffers that are used in flow cytometry. Cytometry protocol for directly staining mouse whole blood. Thus, flow cytometry has become the gold standard method for analysis of complex cell mixtures, from blood, spleen, lymph or bone marrow. Incubate for 30 minutes at room temperature away from light.
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