CD3 MicroBeads, human (92-01-0034)
92-01-0034
Basic information
[Components]
2 mL CD3 MicroBeads, human: MicroBeads conjugated to monoclonal anti-human CD3 antibodies (isotype: mouse IgG2a).
[Size]
For 109 total cells, up to 100 separations.
[Product format]
CD3 MicroBeads are supplied as a suspension containing stabilizer and 0.05% sodium azide.
[Storage]
Store protected from light at 2 - 8 °C. Do not freeze. The expiration date is indicated on the vial labels.
[Reagent and instrument requirements]
● Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA. Keep buffer cold (2−8 °C).
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum or fetal calf serum. Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
● MACS Columns and MACS Separators: CD3+ cells can be enriched by using MS, LS or XS Columns (positive selection). CD3 MicroBeads can be used for depletion of CD3+ cells on LD, CS or D Columns.
Positive selection or depletion can also be performed by using the autoMACS Separator.
Protocol
[Sample preparation]
When working with anticoagulated peripheral blood or buffy coat, peripheral blood mononuclear cells (PBMCs) should be isolated by density gradient centrifugation.
▲ Note: Remove platelets after density gradient separation: resuspend cell pellet in buffer and centrifuge at 200× g for 10−15 minutes at 20 °C. Carefully remove supernatant. Repeat washing step and carefully remove supernatant.
When working with tissues, prepare a single-cell suspension by a standard preparation method.
▲ Note: Dead cells may bind non-specifically to MicroBeads. To remove dead cells, we recommend using density gradient centrifugation or the Dead Cell Removal Kit.
[Magnetic labeling]
▲ Work fast, keep the cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.
▲ Volumes for magnetic labeling given below are for up to 107 total cells. When working with fewer than 107 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2× 107 total cells, use twice the volume of all indicated reagent volumes and total volumes).
▲ For optimal performance it is important to obtain a single-cell suspension before magnetic separation. Pass cells through 30 µm nylon mesh (Pre-Separation Filters) to remove cell clumps which may clog the column.
1. Determine cell number.
2. Centrifuge cell suspension at 300× g for 10 minutes. Pipette off supernatant completely.
3. Resuspend cell pellet in 80 µL of buffer per 107 total cells.
4. Add 20 µL of CD3 MicroBeads per 107 total cells.
5. Mix well and incubate for 15 minutes at 2−8 °C.
▲ Note: Working on ice may require increased incubation times. Higher temperatures and/or longer incubation times lead to non-specific cell labeling.
6. (Optional) Add staining antibodies, e.g. add 10 µL of CD3-FITC and incubate for 5 minutes at 2−8 °C.
7. Wash cells by adding 1−2 mL of buffer per 107 cells and centrifuge at 300× g for 10 minutes. Pipette off supernatant completely.
8. Resuspend up to 108 cells in 500 µL of buffer.
▲ Note: For higher cell numbers, scale up buffer volume accordingly.
▲ Note: For depletion with LD Columns, resuspend up to 1.25× 108 cells in 500 µL of buffer.
9. Proceed to magnetic separation.
[Magnetic separation]
▲ Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD3+ cells.
Magnetic separation with MS or LS Columns
1. Place column in the magnetic field of a suitable MACS Separator.
2. Prepare column by rinsing with appropriate amount of buffer:
MS: 500 µL LS: 3 mL
3. Apply cell suspension onto the column.
4. Collect unlabeled cells which pass through and wash column with appropriate amount of buffer. Perform washing steps by adding buffer three times, each time once the column reservoir is empty.
MS: 3× 500 µL LS: 3× 3 mL
Collect total effluent. This is the unlabeled cell fraction.
5. Remove column from the separator and place it on a suitable collection tube.
6. Pipette appropriate amount of buffer onto the column. Immediately flush out fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column.
MS: 1 mL LS: 5 mL
▲ Note: To increase the purity of the magnetically labeled fraction, it can be passed over a new, freshly prepared column.
Magnetic separation with XS Columns
For instructions on the column assembly and the separation, refer to the "XS Column data sheet".
Depletion with LD Columns
1. Place LD Column in the magnetic field of a suitable MACS Separator.
2. Prepare column by rinsing with 2 mL of buffer.
3. Apply cell suspension onto the column.
4. Collect unlabeled cells which pass through and wash column with 2× 1 mL of buffer. Collect total effluent. This is the unlabeled cell fraction.
Depletion with CS Columns
1. Assemble CS Column and place it in the magnetic field of a suitable MACS Separator.
2. Prepare column by filling and rinsing with 60 mL of buffer. Attach a 22G flow resistor to the 3-way-stopcock of the assembled column.
3. Apply cell suspension onto the column.
4. Collect unlabeled cells which pass through and wash column with 30 mL buffer from the top.
Collect total effluent. This is the unlabeled cell fraction.
Magnetic separation with the autoMACS Separator
▲ Refer to the "autoMACS User Manual" for instructions on how to use the autoMACS Separator.
1. Prepare and prime autoMACS Separator.
2. Place tube containing the magnetically labeled cells in the autoMACS Separator. For a standard separation, choose following separation programs:
Positive selection: "Possel"
Depletion: "Depletes"
▲ Note: Program choice depends on the isolation strategy, the strength of magnetic labeling and the frequency of magnetically labeled cells. For details see autoMACS User Manual: "autoMACS Cell Separation Programs".
3. When using the program "Possel", collect positive fraction (outlet port "pos1"). This is the
purified CD3+ cell fraction.
When using the program "Depletes", collect unlabeled fraction (outlet port "neg1"). This is the CD3- cell fraction.
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