CD25 Nanobeads Kit, mouse (RUO)

Catalog Number: S0K0033 Reactivity: Mouse Conjugation: Brand: Starter

Price:

$500 USD

Size: 50T 100T

Quantity:

For shipping services or bulk orders, you may request a quotation.

Secure checkout with

View full details

Product Details

Product Specification

Format	(1)Dextran-coated iron oxide nanomagnetic beads coupled with anti-PE monoclonal antibodies are stored in a PBS buffer system containing BSA, Poloxamer 188, and NaN.₃In the PBS buffer system. (2)Phycoerythrin (PE)-labeled mouse anti-CD25 monoclonal antibody is stored in a PBS buffer system containing BSA and Proclin300.
Capacity	50T (0.5 mL magnetic beads & 0.1 mL antibody): Suitable for 5 × 10⁸Total cell count: up to 50 sorting runs can be performed. 100T (1 mL magnetic beads & 0.2 mL antibody): Suitable for 1 × 10⁹Total cell count: up to 100 sorting runs can be performed.
Principle of Separation	First, the cells are stained with a mouse anti-CD25 antibody labeled with phycoerythrin (PE), and then the PE‑CD25‑labeled cells are magnetically labeled using Anti‑PE Nanobeads. After labeling is complete, the cell suspension is loaded into a separation column placed within the sorter's magnetic field. The magnetically labeled CD25‑positive cells are retained in the column, while the unlabeled cells flow through the column, constituting the negative fraction. Once the column is removed from the magnetic field, the CD25‑positive cells that were captured by the magnetic beads are eluted, yielding the positive fraction.
Applications in cell sorting	Enrichment or depletion of CD25-positive cells in mice.
Reactivity	Mouse
Magnetic Bead Size	20 nm
Separation Method	Column-Based
Selection Strategy	Positive Selection
Stability & Storage	Store protected from light at 2–8°C; avoid freezing.

Background

CD25, also known as the interleukin-2 receptor alpha chain (IL-2Rα), is expressed in the early stages of thymic T cell development (CD4^-CD8^-); peripheral resting conventional T cells exhibit low CD25 expression, while CD25 is upregulated upon activation of T and B cells, with relatively low expression on activated monocytes. When present alone, CD25 has low affinity for IL-2; however, when it forms a trimeric complex—IL-2Rαβγ—with the β chain (CD122) and the γ chain (CD132)—it creates a high-affinity IL-2 receptor complex that mediates IL-2 signaling.The CD4⁺CD25⁺ cell population includes Foxp3⁺ regulatory T cells (Treg).

Protocol

Note: This kit does not include the sorting buffer; it must be provided separately. The corresponding Stater product number is S0D3018.

Taking SSeparation Columnas an example

	Steps	Operating Procedure	Dosage / Time
Magnetic Labeling	1	Grind a small tissue sample containing CD25-positive cells on a 40 μm sterile filter to prepare a single-cell suspension. After red blood cell lysis, perform live cell counting. Note:It is recommended to count cells after staining with AO/PI.	—
	2	Centrifuge the cell suspension at 400×g for 7 minutes, and completely discard the supernatant.	400×g, 7 min
	3	Resuspend every 1 × 10⁷ cells in 98 μL of buffer. Note:The volume of buffer should be adjusted proportionally according to the sample volume; (e.g., resuspend 2 × 10⁷ cells in 196 μL of sorting buffer).	Resuspend the cells
	4	Add 2 μL of PE-CD25 antibody to label the cells, and mix the cell suspension thoroughly, avoiding excessive bubble formation during mixing. Note:When the cell number increases, proportionally increase the volume of all reagents accordingly.	Add 2 μL of PE-CD25 antibody.
	5	Mix thoroughly and incubate in the dark at 2–8°C for 10 minutes.	Incubate for 10 minutes.
	6	Wash each 1 × 10⁷ cells with 1 mL of buffer, centrifuge at 400 × g for 7 minutes, and completely discard the supernatant.	Add 1 mL of buffer to 1 × 10⁷ cells.
	7	Resuspend each 1 × 10⁷ cells in 90 μL of buffer. Note:Adjust the volume of the buffer proportionally according to the sample volume; (e.g., resuspend 2 × 10⁷ cells in 180 μL of sorting buffer).	Resuspend the cells.
	8	Add 10 μL of Anti-PE Nanobeads per 1 × 10⁷ cells.,And mix the cell suspension thoroughly, avoiding excessive bubble formation during mixing. Note:When the number of cells increases, proportionally increase the volume of all reagents (e.g., for 2 × 10⁷ cells, add 20 μL of magnetic beads).	For 1 × 10⁷ cells, add 10 μL.Anti-PE Nanobeads
	9	Mix thoroughly and incubate in the dark at 2–8°C for 15 minutes.	Incubate for 15 minutes.
	10	Resuspend the cells in 500 μL of sorting buffer. Note:500 μL of buffer can resuspend up to 1 × 10⁸ cells.Cells	Resuspend the cells.
Magnetic separation.	11	Place the S-shaped separation column into the corresponding magnetic separator's magnetic field, and rinse the column with 500 μL of buffer.	Rinse the separation column.
	12	Load the cell suspension onto the separation column and collect the flow-through (unlabeled cells).	—
	13	Wash the column with buffer three times, 500 μL each time, and add the next wash only after the liquid in the column has completely drained.	3 × 500 μL buffer
	14	Combine all eluates into the negative fraction; remove the separation column from the magnetic field and place it over a collection tube.	—
	15	Add 1 mL of buffer to the column and quickly and firmly push down with the provided plunger to elute the magnetically labeled positive cells. Note:For higher purity, you can use a new separation column to repeat the magnetic separation once on the eluted positive cells.	Collect the target cells.

In LSeparation Columnfor example

	Steps	Operating method	Dosage / Time
Magnetic labeling	1	Grind a small tissue sample containing CD25-positive cells through a 40 μm sterile filter to prepare a single-cell suspension. After red blood cell lysis, perform live cell counting. Note:It is recommended to count cells after staining with AO/PI.	—
	2	Centrifuge the cell suspension at 400×g for 7 minutes, and completely aspirate the supernatant.	400×g, 7 min
	3	Resuspend every 1×10⁷ cells in 98 μL of buffer. Note:Adjust the volume of the buffer proportionally according to the sample volume; (e.g., resuspend 2×10⁷ cells in 196 μL of sorting buffer).	Resuspend the cells.
	4	Add 2 μL of PE-CD25 antibody to label the cells, mix the cell suspension thoroughly, avoiding excessive bubble formation during mixing. Note:When the cell number increases, increase the volumes of all reagents proportionally.	Add 2 μL of PE-CD25 antibody.
	5	Mix thoroughly and incubate in the dark at 2–8°C for 10 minutes.	Incubate for 10 minutes.
	6	Wash every 1×10⁷ cells with 1 mL of buffer, centrifuge at 400×g for 7 minutes, and completely aspirate the supernatant.	1 × 10⁷ cells plus 1 mL of buffer
	7	Resuspend every 1 × 10⁷ cells in 90 μL of buffer Note:Adjust the volume of buffer proportionally according to the sample volume (e.g., resuspend 2 × 10⁷ cells in 180 μL of sorting buffer)	Resuspend the cells
	8	Add 10 μL of Anti-PE Nanobeads per 1 × 10⁷ cells,and mix the cell suspension; avoid generating excessive bubbles during mixing Note:When the number of cells increases, increase the volumes of all reagents proportionally (e.g., add 20 μL of magnetic beads for 2 × 10⁷ cells)	1 × 10⁷ cells plus 10 μLAnti-PE Nanobeads
	9	Mix thoroughly and incubate in the dark at 2–8°C for 15 minutes	Incubate for 15 minutes
	10	Resuspend the cells in 1 mL of sorting buffer Note:Resuspend up to 1×10⁸ cells in 500 μL bufferCells	Resuspend cells
Magnetic separation	12	Place the L-shaped separation column into the magnetic field of the corresponding magnetic separator, and rinse the column with 3 mL of buffer	Rinse the separation column
	12	Load the cell suspension onto the separation column and collect the flow-through (unlabeled cells)	—
	13	Wash the column with buffer three times, 3 mL each time; add the next wash only after the liquid in the column has completely drained	3 × 3 mL buffer
	14	Combine all flow-through fractions as the negative fraction; remove the separation column from the magnetic field and place it over a collection tube	—
	15	Add 5 mL of buffer to the column, and use the provided plunger to quickly and firmly push down to elute the magnetically labeled positive cells Note: For higher purity, you can repeat the magnetic separation once on the eluted positive cells using a new separation column	Collect the target cells

Picture

Validation Data

Use the CD25 Nanobeads Kit, mouse (RUO), S or L Separation Column, and a magnetic separator to enrich or deplete CD25+ cells from C57 mouse spleen cells. Subsequently, perform fluorescent staining using PE Rat Anti-Mouse CD25 Antibody (S-R441) (SDT cat: S0B8011), and gate on the live cell population for analysis.

Item added to your cart

CD25 Nanobeads Kit, mouse (RUO)