美天旎磁珠

美天旎磁珠：CD3 MicroBeads, human  CD3磁珠

CD3在所有T细胞和CD56上表达+NKT细胞,与T细胞受体有关。70-80％的人外周血淋巴细胞和65-85％的胸腺细胞是CD3+。由CD3 MicroBeads识别的表位位于CD3ε链上。由MACS分离的T细胞®技术已被用于各种研究,例如关于T细胞细胞毒性,T细胞活化,HIV感染性,信号转导和表面标志物表达。

Index

1.Description

1.1 Principle of MACS® separation

1.2 Background and product applications

1.3 Reagent and instrument requirements

2.Protocol

2.1 Sample preparation

2.2 Magnetic labeling

2.3 Magnetic separation

3.Example of a separation using CD3 MicroBeads

4.References

1.  Description

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 suspensioncontaining stabilizer and 0.05% sodium azide.

Storage Store protected from light at 4 − 8 °C.Do not freeze.Theexpiration date is indicated on the vial label.

1.1  Principle of MACS® separation

First, the CD3+ cells are magnetically labeled with CD3 MicroBeads.Then the cell suspension is loaded onto a MACS® Column which is placed in the magnetic field of a MACS Separator.The magnetically labeled CD3+ cells are retained on the column.The unlabeled cells run through and this cell fraction is depleted of CD3+ cells.After removal of the column from the magnetic field, the magnetically retained CD3+ cells can be eluted as the positively selected cell fraction.

1.2  Background and product applications

CD3 is expressed on all T cells and is associated with the T cell receptor.70–80% of human peripheral blood lymphocytes and 65–85% of thymocytes are CD3 +.The epitope recognized by CD3 MicroBeads is located on the CD3ε chain.

Examples of applications

● CD3 MicroBeads were used for the positive selection1,2,3 or depletion4,5 of T cells from peripheral blood, bronchial lavage, cell culture or various tissues such as lymphoid, nasal and tumor tissue.

● T cells isolated by MACS Technology have been used for various studies, e.g.on T cell cytotoxicity, T cell activation1, HIV infectivity2, signal transduction and surface marker expression.

1.3  Reagent and instrument requirements

● Buffer (degassed): Prepare a solution containing PBS (phosphate buffered saline) pH 7.2, 0.5% BSA and 2 mM EDTA by diluting MACS BSA Stock Solution (# 130-091-376) 1:20 with autoMACS™ Rinsing Solution (# 130-091-222).Keep buffer cold (4−8 °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.

Column max.number max.number Separator

of labeled cells of total cells

Positive selection

MS 107 2×108 MiniMACS, OctoMACS,

VarioMACS, SuperMACS

LS 108 2×109 MidiMACS, QuadroMACS,

VarioMACS, SuperMACS

XS 109 2×1010 SuperMACS

Depletion

LD 108 5×108 MidiMACS, QuadroMACS,

VarioMACS, SuperMACS

CS 2×108 VarioMACS, SuperMACS

D 109 SuperMACS

Positive selection or depletion

autoMACS 2×108 4×109 autoMACS

▲ Note: Column adapters are required to insert certain columns into VarioMACS™ Separator or SuperMACS™ Separator.For details, see MACS Separator data sheets.

● (Optional) Fluorochrome-conjugated CD3 antibody for flow-cytometric analysis, e.g.CD3-FITC (# 130-080-401), CD3-PE (# 130-091-374), or CD3-APC (# 130-091-373).

● (Optional) PI (propidium iodide) or 7-AAD for flow- cytometric exclusion of dead cells.

● (Optional) Pre-Separation Filters (# 130-041-407) to remove cell clumps.

2.  Protocol

2.1  Sample preparation 2.3  Magnetic separation

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 MACS MicroBeads.To remove dead cells, we recommend using density gradient centrifugation or the Dead Cell Removal Kit (# 130-090-101).

2.2  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 # 130-041-407) 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 4−8 °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 (# 130-080-401), and incubate for 5 minutes at 4−8 °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 (2.3).

▲ Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD3+ cells (see table in section 1.3).

Magnetic separation with MS or LS Columns

1.Place column in the magnetic field of a suitable MACS Separator (see "Column data sheets").

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 (see "LD Column data sheet").

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 (see "CS Column data sheet").

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 (see "CS Column data sheet").

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.

Depletion with D Columns

For instructions on column assembly and separation, refer to the "D Column data sheet".

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use.

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.

3.  Example of a separation using CD3 MicroBeads

Separation of PBMCs using CD3 MicroBeads and a MiniMACS™ Separator with an MS Column.The cells are fluorescently stained with CD3-FITC (# 130-080-401).Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.

PBMCs before separation

Relative cell number

CD3-FITC

CD3+ cells

Relative cell number

CD3-FITC

4.  References

1.Pitti, RM!Marsters, SA!Lawrence, DA!Roy, M!Kischkel, FC!Dowd, P!Huang, A!Donahue, CJ!Sherwood, SW!Baldwin, DT!Godowski, PJ!Wood, WI!Gurney, AL!Hillan, KJ!Cohen, RL!Goddard, AD!Botstein, D!Ashkenazi, A (1998) Genomic Amplification of a Decoy Receptor for Fas Ligand in Lung and Colon Cancer.Nature 396: 699–703.[517]

2.Heath, S!Tew, J!Szakal, A!Burton, G (1995) Follicular Dendritic Cells and Human Immunodeficiency Virus Infectivity.Nature 377: 740–744.[177]

3.Lorenzen, DR!Düx, F!Wölk, U!Tsirpouchtsidis, A!Haas, G!Meyer, TF (1999) Immunoglobulin A1 Protease, an Exoenzyme of Pathogenic Neisseriae, Is a Potent Inducer of Proinflammatory Cytokines.J.Exp.Med.190: 1049–1058.[882]

4.Heidenreich, F!Jovin, T (1996) Synthesis of anti-acetylcholine receptor antibodies by CD5- B cells from peripheral blood of myasthenia gravis patients.J.Neurol.243: 57–62.[441]

5.Klein, U!Küppers, R!Rajewsky, K (1994) Variable Region Gene Analysis of B Cell Subsets Derived from a 4-year-old Child: Somatically Mutated Memory B Cells Accumulate in the Periperal Blood Already at Young Age.J.Exp.Med.180: 1383– 1393.[75]

Warnings

Reagents contain sodium azide.Under acidic conditions sodium azide yields hydrazoic acid, which is extremely toxic.Azide compounds should be diluted with running water before discarding.These precautions are recommended to avoid deposits in plumbing where explosive conditions may develop.

Warranty

The products sold hereunder are warranted only to be free from defects in workmanship and material at the time of delivery to the customer.MILTENYI BIOTEC GmbH makes no warranty or representation, either expressed or implied, with respect to the fitness of a product for a particular purpose.There are no warranties, expressed or implied, which extend beyond the technical specifications of the products.MILTENYI BIOTEC GmbH's liability is limited to either replacement of the products or refund of the purchase price.MILTENYI BIOTEC GmbH is not liable for any property damage, personal injury or economic loss caused by the product.

Ficoll-Paque is a trademark of GE Healthcare companies.

MACS is a registered trademark of Miltenyi Biotec GmbH.

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use.

美天旎磁珠：CD3 MicroBeads, human  CD3磁珠