Flow type absolute counting microspheres

NegotiableUpdate on 05/06

Model

Nature of the Manufacturer: Producers

Product Category

Place of Origin

Online Consult

Overview

Flow cytometry absolute counting microspheres are now widely used in cell counting experiments and disease progression research, especially in the field of stem cell research.

Product Details

Flow type absolute counting microspheres

Product Description

description

Flow cytometry cell counting analysis can quickly quantify the characteristics of cells, and absolute cell counting microspheres are now widely used in cell counting experiments and disease progression research, especially in the field of stem cell research. The single packaging bottle of this product contains fluorescent particles precisely stained with fluorescent dyes, which have the best fluorescence intensity and a wide emission spectrum in multiple channels (FITC, PE, PE-Cy5). The hydrophilic particle surface can eliminate the generation of dimers, ensuring the accuracy of counting verified by the analysis process and the reliability of absolute counting in flow cytometry.
There are generally two methods for obtaining absolute cell counts: one is to combine the cell concentration detected from the cell analysis instrument with the cell population data analyzed by flow cytometry (multi platform detection), and the other is to add internal microsphere counting standards to the flow cytometry sample (single platform detection). The single platform detection method is very simple, avoiding inter laboratory differences and making it more accurate. The use of fluorescent reference microspheres for absolute cell counting is currently widely recognized as one of the flow cytometer single platform absolute counting methods by clinical and scientific researchers.

application

Absolute count of T lymphocytes, CD34+hematopoietic stem cells, reticulocytes, circulating tumor cells, etc

concentration

6.4x107Particles/ml ± 5%

Usage

Sample requirements:
1. Collect a sample of 200uL or more using heparin anticoagulant for blood collection.
2. The sample should be stored at room temperature and avoid shaking. It should be used within 24 hours after blood collection.
3. After staining, the sample should be stored in the dark at 2-8 ℃ and tested on the machine within 24 hours.
4. If the sample is contaminated with microorganisms, has poor lipid, coagulation, and cell viability, it should be avoided from use unless the sample is irreplaceable. Please indicate this in the result report.

Materials to be self provided:
1. Hemolytic agent j is used to separate blood cells
2. Flow cytometry antibody reagent
3. Heparin anticoagulant blood collection
4. The range is suitable for pipettes with 20uL, 100uL, and 1mL, as well as disposable pipettes
5. Vortex oscillator
6. Flow cytometer

Operation steps:
1. Use reverse pipetting technique to extract 50uL of mixed sample and add it to the bottom of the absolute counting tube. The front end of the pipette should avoid contact with the bottom of the tube to prevent blood from touching the upper part of the tube wall.
2. Add a certain amount of flow cytometric antibody reagent at the bottom of the flow cytometer.
3. Cover the tube cap, gently vortex and mix for about 3 seconds, and react at room temperature in the dark for 25 minutes.
4. Add 450uL of blood cells into the tube for hemolysis, cover the tube cap, gently vortex and mix for about 15 seconds, and react at room temperature in the dark for 10 minutes. No washing required, awaiting inspection.
5. Gently vortex and mix for about 10 seconds, then test on the machine.
The following demonstrates the analysis method using normal adult peripheral blood samples and CD3-FITC/CD8-PE/CD45 PerCP/CD4 APC fluorescent monoclonal antibody kit (flow cytometry method) as an example.
Firstly, establish a CD45/SSC scatter plot, where the lymphocyte population shows high expression of CD45 and low expression of SSC, circled with irregular gates, as shown in Figure 1:

Figure 1: Circle lymphocyte gates in CD45/SSC scatter plot

Secondly, select to display cells within the lymphocyte phylum and set up a CD3/SSC scatter plot. Circle CD3+, as shown in Figure 2.

Figure 2: CD3+lymphocytes circled in the CD3/SSC scatter plot

Select to display cells within the lymphocyte phylum and set a four quadrant threshold for CD3/CD4 scatter plot. CD3 and CD4 double positive cells are CD4+T lymphocytes, as shown in Figure 3.

Figure 3: CD3/CD4 scatter plot with four quadrant gates set

Finally, select to display all cells, set up a CD3/CD8 scatter plot, and circle the absolute count microspheres, as shown in Figure 4.

Figure 4: CD3/CD8 scatter plot circled with absolute counting microspheres

If automatic software for absolute counting is not used, the following equation can be used for manual counting:
Absolute number of lymphoid subpopulations (cells/uL)=(number of lymphoid subpopulation cells within the region x microsphere marker value)/(absolute count of microsphere within the region x sample volume)

Storage/Preservation Methods

Store at 2-30 ℃, do not freeze, valid for 5 years.

technical indicators

component	Multi fluorescence stained polystyrene microspheres
Microsphere size	5 um
color	Green fluorescence (488/525 nm)
	Red fluorescence (535/575 nm)
	Near infrared fluorescence (635/660 nm)
concentration	6.4×107Particles/ml ± 5%
density	1.05 g/cm³
Once average	CV＜3%
additive	0.05% Tween-20 dispersant/surfactant, 2mM preservative

Precautions

1. Do not freeze.
2. Ultrasound oscillation for 10 seconds before use is effective.

research field
research field	Drug DiscoverySmall Molecule DrugLead Compound Discovery

Flow type absolute counting microspheresKind reminder: This product is only for scientific research experiments and does not support clinical research