CD3ε Nanobeads Kit, mouse (RUO)

Catalog Number: S0K0015 Reactivity: Mouse Conjugation: Brand: Starter

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$335 USD

Size: 1mL 2mL

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Product Details

Product Specification

Format	Biotinylated anti-mouse CD3ε monoclonal antibody and streptavidin-conjugated nanomagnetic beads, in a PBS buffer system containing BSA and Poloxamer 188.
Capacity	1 mL magnetic beads: Suitable for a total cell count of 5×10⁸, allowing for up to 50 sorting runs. 2 mL magnetic beads: Suitable for a total cell count of 1×10⁹, allowing for up to 100 sorting runs.
Principle of Separation	First, CD3ε+ cells are labeled with a CD3ε-biotin–conjugated primary antibody. Subsequently, the cells are magnetically labeled using biotin-conjugated nanomagnetic beads. The cell suspension is then placed in a separation column within a magnetic field. The CD3ε+ cells that have been labeled with the magnetic beads remain retained in the column, while the unlabeled cells flow through; thus, CD3ε+ cells are separated from the other cell populations.
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; do not freeze.

Background

CD3ε is a transmembrane protein with a molecular weight of 20 kDa and serves as a subunit of the T-cell receptor (TCR) complex. Together with the γ and δ subunits of CD3, it plays a crucial role in the assembly and expression of the TCR complex. CD3 is expressed on the surface of thymocytes, mature T lymphocytes, and natural killer T cells (NKT cells). Using the CD3ε nanosphere kit, mouse T lymphocytes and NKT cells can be depleted from various tissues, including the spleen, thymus, and lymph nodes.

Protocol

Note: This kit does not include the sorting buffer; please prepare it yourself. The corresponding Stater catalog number isS0D3018

Taking S Separation Column as an example

	Steps	Operating method	Dosage / Time
Magnetic labeling	1	Count the total number of cells	—
	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 95 μL of buffer Note:The buffer must be freshly prepared and used immediately	Resuspending cells
	4	Add 5 μL of CD3ε-Biotin per 1×10⁷ cells Note:When the cell quantity increases, proportionally increase the volume of all reagents accordingly	5 μL per 1×10⁷ cells
	5	Mix thoroughly and incubate in the dark at 2–8 ℃ for 10 minutes	10 min
	6	Wash 1×10⁷ cells with 1 mL of buffer, centrifuge at 400×g for 7 minutes, and completely discard the supernatant.	Add 1 mL to 1×10⁷ cells, centrifuge at 400×g for 7 min.
	7	Resuspend 1×10⁷ cells in 80 μL of buffer.	Resuspend the cells.
	8	Add 20 μL of Streptavidin Beads to 1×10⁷ cells. Note: When the cell number increases, proportionally increase the volume of all reagents accordingly.	Add 20 μL to 1×10⁷ cells.
	9	Mix thoroughly, incubate in the dark at 2–8 ℃ for 15 minutes.	15 min
	10	Resuspend up to 1.25×10⁸ cells in 500 μL of buffer.	Resuspend the cells.
Magnetic separation.	11	Place the S-shaped separation column into the magnetic field of the corresponding magnetic separator.	—
	12	Rinse the separation column with buffer.	500 μL buffer
	13	Load the cell suspension onto the sorting column and collect the flow-through (unlabeled cells)	—
	14	Wash the column with buffer three times, adding the next wash only after the reservoir has completely drained	3 × 500 μL buffer
	15	Combine all flow-through fractions into the negative fraction; remove the sorting column from the magnetic field and place it over a collection tube	—
	16	Add buffer to the column and use the provided plunger to quickly and firmly push down, eluting the magnetically labeled positive cells NoteIf higher purity is required, repeat the separation once using a new column	1 mL buffer

Taking the D Separation Column as an example

	Steps	Operating method	Dosage / Time
Magnetic labeling	1	Count the total number of cells	—
	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 95 μL of buffer Note:The buffer must be freshly prepared and used immediately	Resuspend the cells
	4	Add 5 μL of CD3ε-Biotin to every 1×10⁷ cells Note:When the cell number increases, proportionally increase the volume of all reagents accordingly	For 1×10⁷ cells, add 5 μL
	5	Mix thoroughly, incubate at 2–8 ℃ in the dark for 10 minutes	10 min
	6	Wash every 1×10⁷ cells with 1 mL of buffer, centrifuge at 400×g for 7 minutes, and completely discard the supernatant	For 1×10⁷ cells, add 1 mL, centrifuge at 400×g for 7 min
	7	Resuspend every 1×10⁷ cells in 80 μL of buffer	Resuspend the cells
	8	Add 20 μL of Streptavidin Beads to every 1×10⁷ cells Note: When the cell number increases, all reagent volumes should be increased proportionally at the same time.	Add 20 μL to 1×10⁷ cells
	9	Mix thoroughly and incubate in the dark at 2–8 ℃ for 15 min	15 min
	10	Resuspend up to 1.25×10⁸ cells in 500 μL of buffer	Resuspend the cells
Magnetic separation	11	Place the D-type separation column into the magnetic field of the corresponding magnetic separator.	—
	12	Wash the separation column with buffer.	3 mL of buffer
	13	Load the cell suspension onto the separation column and collect the flow-through (unlabeled cells).	—
	14	Wash the column with buffer three times, waiting for the reservoir to completely empty before adding the next wash.	3 × 3 mL of buffer
	15	Combine all flow-through fractions into the negative fraction; remove the separation column from the magnetic field and place it over the collection tube.	—
	16	Add buffer to the column, and use the provided plunger to quickly and firmly push, eluting the magnetically labeled positive cells. Note: For higher purity, replace with a new column and repeat the separation once.	5 mL buffer

Picture

Validation Data

Using the CD3ε Nanobeads Kit, mouse S(D) separation columns, and a sorter, CD3ε-positive cells were isolated from the single-cell suspension. The cells were fluorescently labeled with an anti-CD3ε+ (clone: S-R491) Alexa Fluor® 488 antibody (SDT Cat: S0B5473), and the "live cell+" population was selected.

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CD3ε Nanobeads Kit, mouse (RUO)