Product Introduction

This product is a recombinant protein that can cross-link CD3+ cells with red blood cells in whole blood. Then, CD3+ cells can be directly removed from whole blood using traditional Ficoll density gradient centrifugation. It is convenient and quick, and the amount of blood processed is 40-50ml whole blood (peripheral blood, umbilical cord blood). This product can remove more than 90% of CD3+ cells, providing a powerful tool for basic and clinical research.

Features

• Quick operation, CD3+ cell removal can be completed during density gradient centrifugation.
• High removal rate of target cells, more than 90% of target cells can be removed.
• The purification reagents are mild and have little impact on subsequent experiments.

How to use

• 1. Reconstitution of purified reagents: Take 500 μL of sterile DPBS (sterile water for injection or normal saline can also be used) and add it to the freeze-dried purified reagents, mix well and set aside.
• 2. Centrifugal collection of cells: Take a small amount of blood sample from 40-50 mL of fresh anticoagulated whole blood from the donor for sterile streak test, transfer the remaining blood sample to a 50 ml sterile centrifuge tube, and centrifuge at 1000 g (slow rise 1, slow drop 0) for 15 minutes . Transfer the upper plasma to a new 50 ml sterile centrifuge tube , collect the lower cells and perform purification according to step
• 3. Note: If inactivated plasma is required, the preparation method is as follows : centrifuge the upper plasma collected in step 2 at 2100g (slow rise 8, slow descend 6) for 10 minutes, transfer the supernatant to a new sterile centrifuge tube, inactivate in a 56℃ water bath for 30 minutes, cool in a 4℃ refrigerator for 30 minutes (or place in a -20℃ refrigerator for 15 minutes), and then centrifuge again at 2100g (slow rise 8, slow descend 6) for 15 minutes, transfer the supernatant to a new sterile centrifuge tube, which is the heat-inactivated autologous plasma, and store at 4℃ for later use.
• 4. Preparation of NK cell purification: Dilute the cells collected by centrifugation in step 2 with DPBS or saline at a ratio of 1:1, gently blow and mix, add one tube of purification reagent prepared in step 1 and immediately invert and mix, incubate at room temperature for 20 minutes, and mix 4-5 times during the period (if the liquid volume in this step is large, the cell suspension can be appropriately divided into two 50ml centrifuge tubes, and the corresponding purification reagent can be divided into two parts for use). After incubation, dilute with sterile DPBS or saline at a ratio of 1:1, and gently invert and mix. Take a 50ml sterile centrifuge tube, add Ficoll solution according to the ratio of blood:Ficoll=2:1, and then carefully add blood to the Ficoll layer to prevent the blood from breaking through the Ficoll layer. Centrifuge at 1200g (slow rise 1, slow fall 0) for 20min. Carefully aspirate the white film layer into a 50ml centrifuge tube containing 20ml DPBS for washing, 300g (slow rise 8, slow fall 6) at room temperature for 10min, discard the supernatant after centrifugation, and repeat washing 1-2 times. Resuspend the cells in 5 mL of pre-warmed NK serum-free medium, take a sample and count the cells for subsequent experiments .

Experimental results (FACS)

 

             Figure 1                                       Figure 2

 

                 Figure 3                                          Figure 4