Streptamer® CD3/CD28 Premix for T cell expansion Manual · 2020-06-28 · Human T cells were...

Last date of revision April 2016 Version PR49-0001

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Streptamer® CD3/CD28 Premix for T cell

expansion Manual Easy activation of human T cells with reversible reagents

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

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 http://www.iba-go.com/patents.html or upon inquiry at [email protected] or at IBA GmbH, Rudolf-Wissell-Str. 28, 37079 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 http://www.iba-go.com/patents.html. Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.

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 106 up to 1 x 106 T cells in 1 ml

cell culture medium.


Table 1: Volume recommendations

Type of cell culture 96-well plate 48-well plate 24-well plate

Cell number 5 – 8x104 2 – 5x105 0.5 – 1x106

Seeding volume 100 – 200 µl 0.5 – 1 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 10 – 20 µl

Activate human T cells

4. Resuspend 0.5 – 1 x 106 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 CO2 incubator at 37°C, according to your

experimental setup.

6. Harvest activated T cells and use directly for further analysis.


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 106 cells/ml). If cell medium turns

yellow or cell density is too high, split cultures back to a density of

0.2 – 1 x 106 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.


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. 6-0219-001) 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.


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

6-8000-201 CD3 Fab Streptamer® Isolation Kit, human

for isolation out of 1x10^9 cells with magnetic microbeads (MB); to be used with a permanent magnet





6-5650-065

StrepMan permanent magnet

6-0219-001

D-Biotin Stock Solution

1 ml


5 Data Examples

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 (6-8000-201) 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 (6-8000-201) were cultured with (A) or without (B) CD3/CD28 Streptamers® for T cell expansion.

Expansion of human CD3+ Tc

0 3 6 10 130

20

40

60

80

Days

Fo

ld e

xp

an

sio

n


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 (6-8000-201) 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.

CD25-PE

CD69-APC

6,5% 44,3% 94,7%

2,7% 64,7% 45,0%

day0 day3 day6


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 (6-8000-201), 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.

CD4+

CD8+

#

CFSE

unstimulated CD3/CD28 Streptamer


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.

IBA Headquarter IBA GmbH Rudolf-Wissell-Str. 28 37079 Goettingen Germany Tel: +49 (0) 551-50672-0 Fax: +49 (0) 551-50672-181 E-mail: [email protected]

Streptamer® CD3/CD28 Premix for T cell expansion Manual · 2020-06-28 · Human T cells were...

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