Streptamer CD3/CD28 Premix for T cell expansion Manual Easy activation of human T cells with reversible reagents

Transcription

1 Streptamer CD3/CD28 Premix for T cell expansion Manual Easy activation of human T cells with reversible reagents Last date of revision April 2016 Version PR

2 For research use only Important licensing information The technologies Streptamer and Strep-Tactin are covered by intellectual property (IP) rights. On completion of the sale of a respective product IBA grants a Limited Use Label License to purchaser. IP rights and Limited Use Label Licenses are further identified at or upon inquiry at info@ibago.com or at IBA GmbH, Rudolf-Wissell-Str. 28, Göttingen, Germany. By use of a respective product the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses. All products are for research use only. CAUTION: Not intended for human or animal diagnostic or therapeutic uses. Trademark information The owners of trademarks marked by or TM are identified at Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. 2 Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents

3 1 Easy activation of human T cells with reversible Streptamers for T cell expansion Scheme of activation of T cells and subsequent biotin induced dissociation of the reagents to terminate the process accurately defined. 2 Protocol The Streptamers for T cell expansion have been developed for easy activation of human T cells without preparing antigen-presenting cells (APCs) or antigen. Preparation 1. Isolate primary T cells or subsets of interest from blood or buffy coat. 2. Prepare cell culture medium. 3. Optimal cell number and density have to be titrated for each individual experimental approach. Generally we recommend a range of 0.2 x 10 6 up to 1 x 10 6 T cells in 1 ml cell culture medium. Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents 3

4 Table 1: Volume recommendations Type of cell culture 96-well plate 48-well plate 24-well plate Cell number 5 8x x x10 6 Seeding volume µl ml 1 2 ml Streptamer CD3/CD28 Premix for T cell expansion 3 µl 15 µl 30 µl 100 mm D-Biotin 1 2 µl 5 10 µl µl Activate human T cells 4. Resuspend x 10 6 purified T cells in 1 2 ml prepared cell culture medium in a 24-well plate. Add 30 µl Streptamer CD3/CD28 Premix for T cell expansion and resuspend gently (see Table 1 if other cell numbers used). 5. Incubate cell suspension in a humidified CO 2 incubator at 37 C, according to your experimental setup. 6. Harvest activated T cells and use directly for further analysis. 4 Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents

5 Expand human T cells 7. For further expansion: Examine culture daily regarding cell size, shape and cluster formation (using a microscope). Count the cells (at least every two or three days) to evaluate cell density (should not exceed 2.5 x 10 6 cells/ml). If cell medium turns yellow or cell density is too high, split cultures back to a density of x 10 6 cells/ml into a new plate of appropriate size. 8. Restimulation of the cells might be necessary after a couple of days in culture (signs of exhaustion typically after 7 10 days). Repeat therefore from step 4. Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents 5

6 D-Biotin dissociation of Streptamers for T cell expansion reagents To terminate T cell stimulation or release of Streptamers for T cell expansion reagents, add D-Biotin directly to the cell culture medium. Removal of all reagents can be performed by harvesting cells and subsequent washing. 9. Add the appropriate amount of D-Biotin stock solution (100 mm; Cat. no ) directly to the culture medium containing the activated T cells. Dissociation of Streptamers for T cell expansion reagents is achieved at a final D-Biotin concentration of 1 mm, obtained by a dilution of 1:100 (i.e. add 10 µl 100 mm D-Biotin per 1 ml cell culture). 10. Incubate cell suspension for 30 min at room temperature. 11. Harvest cells by thoroughly resuspending cells and transfer to a 15 ml reaction tube. Add an appropriate amount (i.e. 10 ml) of culture medium for dissociation of Streptamers for T cell expansion reagents. Incubate for further 10 min and collect cells by centrifugation at 300 x g for 6 10 min. 12. For the complete removal of all expansion reagents discard supernatant and carefully resuspend cells in 15 ml cell culture medium. Incubate for 10 min and repeat centrifugation at 300 x g for 6-10 min. 13. Discard supernatant and use the expanded/activated T cells directly for further analysis. 6 Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents

7 3 Use and storage of Streptamer CD3/CD28 Premix for T cell expansion Streptamer CD3/CD28 Premix for T cell expansion is shipped on blue ice and stored at 4 C until use. 4 Related Products Cat.no. Product Name Size CD3 Fab Streptamer Isolation Kit, human for isolation out of 1x10^9 cells with magnetic microbeads (MB); to be used with a permanent magnet CD4 Fab Streptamer Isolation Kit, human CD8 Fab Streptamer Isolation Kit, human for isolation out of 1x10^9 cells with magnetic microbeads (MB); to be used with a permanent magnet for isolation out of 1x10^9 cells with magnetic microbeads (MB); to be used with a permanent magnet StrepMan permanent magnet D-Biotin Stock Solution 1 ml Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents 7

8 Fold expansion 5 Data Examples Expansion of human CD3 + Tc Days Figure 1: T cell expansion after CD3/CD28 stimulation with Streptamers for T cell expansion. Human T cells isolated with the CD3 Fab Streptamer Isolation Kit MB ( ) were cultured for 13 days with the CD3/CD28 Streptamers for T cell expansion. Fold expansion was measured. Figure 2: Microscopy of T cells in culture showing cluster formation on day 3 after stimulation with Streptamers for T cell expansion. Human T cells isolated with the CD3 Fab Streptamer Isolation Kit MB ( ) were cultured with (A) or without (B) CD3/CD28 Streptamers for T cell expansion. 8 Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents

9 day0 day3 day6 6,5% 44,3% 94,7% CD25-PE 2,7% 64,7% 45,0% CD69-APC Figure 3: T cell activation markers CD25 and CD69 after stimulation with CD3/CD28 Streptamers for T cell expansion. Human T cells isolated with the CD3 Fab Streptamer Isolation Kit MB ( ) were cultured for 6 days. Activation markers CD25 and CD69 were accessed in pregated CD3 + T cells using Flow Cytometry. Dead cells were excluded from the analysis using DAPI. Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents 9

10 # CD4 + unstimulated CD3/CD28 Streptamer CD8 + CFSE Figure 4: T cell proliferation after stimulation with CD3/CD28 Streptamers for Tcell expansion. Human T cells were isolated with the CD3 Fab Streptamer Isolation Kit MB ( ), stained with CFSE and cultured in a 48 well plate for 5 days. Proliferation was measured in pregated CD4 + (upper panel) or CD8 + T cells (lower panel) using Flow Cytometry. Dead cells were excluded from the analysis using PI. 10 Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents

11 6 Warranty The product sold hereunder is warranted only to conform to the quantity and contents stated on the label at the time of delivery to the customer. There are no warranties, expressed or implied, that extend beyond the description on the label of the product. IBA s sole liability is limited to either replacement of the products or refund of the purchase price. IBA GmbH is not liable for property damage, personal injury, or economic loss caused by the product. Streptamer CD3/CD28 Premix for T cell expansion - Manual Easy activation of human T cells with reversible reagents 11

12 IBA Headquarter IBA GmbH Rudolf-Wissell-Str Goettingen Germany Tel: +49 (0) Fax: +49 (0)