CytoSinctTM CD3 Nanobeads, human

Cat.No. L00896

Contents

I. Product Description............................................................................................................. 1

II. Product Specification.......................................................................................................... 1

III. Requirement Materials........................................................................................................ 2

IV. Protocol................................................................................................................................... 2

V. Product Example Data......................................................................................................... 4

I. Product Description

The CytoSinctTM CD3 Nanobeads, human are used for separating CD3+ cells from fresh or frozen

peripheral blood mononuclear cells (PBMCs), leukapheresis products or single cell suspension

based on the surface expression of human CD3. The surface of Nanobeads are labeled with antihuman CD3 monoclonal antibody. To begin the isolation, Nanobeads are added to the cells. The

CD3 antibodies preconjugated to the Nanobeads can bind the target cells expressing CD3 on cell

surface. The cells/beads mixed suspension is loaded onto a CytoSinctTM Column which is

effectively magnetized by an external magnetic field from CytoSinctTM Magnet, or other compatible

cell isolation columns and magnets. The Nanobeads-labeled CD3+ cells are retained within the

column and enriched during the wash step when Isolation Buffer is used to flush out the CD3- cells.

After removing magnetic field, the target CD3+ cells can be easily eluted from the column.

II. Product Specification

Cat. No. Name Size Capacity

L00896-0.5 CytoSinctTM CD3 Nanobeads, human 500 μL for up to 5×108

total cells

L00896-1 CytoSinctTM CD3 Nanobeads, human 1 mL for up to 1×109 total cells

Reactivity Human

Product format Bio-degradable matrix coated nanoparticle conjugated with anti-CD3

antibodies supplied in phosphate buffered-saline (PBS), containing

Human Serum Albumin (HSA), pH 7.0-7.4.

Application Positive selection or depletion of CD3+ T cells from leukapheresis, PBMC

or cell cultures. Isolated CD3+ T cells can be used for culture and

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expansion, flow cytometry, T cells functional assays.

Storage Store at 2-8 °C. Do not freeze.

III. Requirement Materials

1. Isolation Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5%

bovine serum albumin (BSA) and 2 mM EDTA.

 Keep Isolation Buffer cold (2−8°C).

 BSA can be replaced by human serum albumin (HSA), human serum or fetal bovine serum

(FBS).

 EDTA can be replaced by sodium citrate.

 PBS containing Ca2+ or Mg2+ is not recommended.

2. Columns and separators:

 For samples containing less than 2×108 total MNCs or less than 107

labeled cells, use

CytoSinctTM gM Column and CytoSinctTM M1 Magnet or CytoSinctTM M8 Magnet, or other

compatible columns and magnets.

 For samples containing less than 2×109

total cells or less than 108

labeled cells, use

CytoSinctTM gL Column and CytoSinctTM L1 Magnet or CytoSinctTM L4 Magnet or other compatible

columns and magnets.

IV. Protocol

All procedures are to be performed at room temperature unless otherwise instructed in this protocol.

1. Prepare Nanobeads

Gently mix the Nanobeads by pipetting for several times.

2. Prepare samples

2.1 Prepare PBMCs.

 When working with anticoagulated peripheral blood, peripheral blood mononuclear cells

(PBMCs) should be isolated by density gradient centrifugation method (for example, using FicollPaqueTM PLUS density gradient media) and washed by Isolation Buffer to remove interfering factors.

 When working with frozen PBMCs, resuscitate frozen PBMCs and then proceed with the

protocol. When dead cells are found to be considerable, apply density gradient centrifugation

method (for example, using Ficoll-PaqueTM PLUS density gradient media) to remove dead cells, or

culture cells in medium overnight before proceeding with this protocol.

2.2 Centrifuge PBMCs at 300×g for 10 minutes at room temperature (15 - 25°C). Aspirate the

supernatant completely. Determine cell number by using a hemocytometer or other suitable

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methods.

3. Magnetic labeling

3.1 Transfer desired number of cells into a new tube and resuspend into single cell suspension at

108 mononuclear cells (MNCs) per 1 mL in Isolation Buffer.

 When working with less than 107 MNCs, use Isolation Buffer volume of 100 µL.

3.2 Add 10 µL Nanobeads for each 100 µL cell suspension of 107

total MNCs.

 When working with higher number of cells, scale up the volume of Nanobeads accordingly

（e.g. for 2×107

total MNCs，use 20 µL Nanobeads.)

 When working with less than 107 MNCs, use the same Nanobeads volume of 10 µL, as that in

107 MNCs.

3.3 Mix the Nanobeads and cells well by gently pipetting or tapping on the bottom of the tube, and

incubate for 15 min at 2 – 8 °C.

3.4 Wash cells once by adding 1-2 mL of Isolation Buffer per 107 MNCs, mix well by gentle

pipetting, and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.

3.5 Resuspend up to 108 cells in 500 µL of Isolation Buffer.

 Scale up the volume of Isolation Buffer accordingly when more than 108 MNCs are to be

processed.

4. Magnetic separation

4.1 Choose an appropriate CytoSinctTM Column and CytoSinctTM Magnet or other compatible

columns and magnet according to the number of total cells and the number of CD3+ cells as

instructed in Section III.

4.2 Assemble the column onto the suitable CytoSinctTM Magnet or other compatible magnet

(please refer to manual of CytoSinctTM Magnet or other compatible magnets for assembly

instructions).

4.3 Rinse the column once with Isolation Buffer (500 µL for CytoSinctTM gM column, 3 mL for

CytoSinctTM gL Column, or other compatible columns) and let the buffer run through it but not run

dry.

4.4 Transfer the cell suspension onto the prepared CytoSinctTM gM or CytoSinctTM gL Column or

other compatible columns using a pipette and collect the unlabeled cells in flow-through.

4.5 Wash the column with Isolation Buffer (500 µL × 3 for CytoSinctTM gM Column, 3 mL × 3 for

CytoSinctTM gL Column, or other compatible columns). Collect unlabeled cells in flow-through with

a suitable tube (for example, a 2 mL or 15 mL conical tube). Repeat the washing step for another

two times. Add new Isolation Buffer when the column stops dripping but not run dry.