大花石上莲Contents
1. Description
1.1 Background information
1.2 Applications
1.3 Reagent and instrument requirements
2. Protocol
2.1 Sample preparation
2.2 T cell activation and expansion
2.3 Immunofluorescent staining
3. Examples of T cell activation and expansion using the
T Cell TransAct™
ttx2基板
1. Description
This product is for research use only.
Components2×2mL T Cell TransAct™, human
Capacity The reagent is sufficient to activate and expand up
to 4×108 enriched T cells or up to 8×108 peripheral
blood mononuclear cells (PBMCs), when used at
recommended titer of 1:100.
Product format Polymeric nanomatrix conjugated to humanized
CD3 and CD28 agonist supplied in phosphate-
buffered-saline (PBS), containing 0.03%
poloxamer 188 as stabilizer, pH 7.3–7.9. Storage Store protected from light at 2−8 °C. Do not
freeze. The expiration date is indicated on the
vial label.
1.1 Background information
The T Cell TransAct has been designed to activate and expand enriched T cell populations or human resting T cells from periphal blood mononuclear cells (PBMCs). T cell expansion is achieved by culturing for up to 14 days. For longer cultivation restimulation after 14 days is necessary.
Polyclonal T cell expansion can be used when increased numbers of T cells are required or when T cells are activated to enable gene modification.
Due to the nanomatrix of the T Cell TransAct, it can be sterile filtered and excess reagent can be removed by simple replacement of supernatant or by a washing step, e.g., centrifugation.
The recommended titers have been found to efficiently stimulate the majority of T cell subsets, however, for special applications it is recommended to experimentally determine the optimal stimulation titer. Over-activation of T cells carries a risk of activation-induced cell death.
The T Cell TransAct has been developed in combination with the TexMACS™ Medium and Human IL-2 IS or Human IL-7 and Human IL-15.1.2 Applications
●The T Cell TransAct is intended for the in vitro stimulation
and expansion of purified T cell populations of, for example,
untouched T cells isolated with the Pan T Cell Isolation K it,
human, as well as of human T cells from hematological cell
populations (e.g. PBMCs).
1.3 Reagent and instrument requirements
● TexMACS Medium, research grade (#
130-097-196) supplemented with Human IL-2 IS, premium grade书立
(# 130-097-744) or Human IL-7, premium grade (# 130-095-361)
and Human IL-15, premium grade (# 130-095-762).
红薯清洗机●Buffer for flow cytometric analysis: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5%
bovine serum albumin (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
(2−8 °C).
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
●Fluorochrome-conjugated antibodies for flow
cytometric analysis, e.g., CD4-VioBlue®, CD8-VioGreen™,
CD25-PE, and CD69-APC. For more information
about fluorochrome-conjugated antibodies refer to
www.miltenyibiotec/antibodies.
●(Optional) Pan T Cell Isolation Kit, human (# 130-096-535)
●(Optional) Propidium Iodide Solution (# 130-093-233) or
7-AAD for flow cytometric exclusion of ded cells.
2. Protocol
▲All steps in the protocol have to be performed under steril conditions.
▲Excess of T Cell TransAct is removed by simple replacement
of supernatant or by a washing step, e.g., centrifugation (at least
10-fold reduction) 2–3 days after initial stimulation. Performing a washing step earlier may result in reduced T cell proliferation.
▲ Activated T cells can be transduced 1–2 days after activation. The optimal virus titer has to be defined before and depends on the viral vector used. The T Cell TransAct can be used in combination with retro- or lenti-viral transduction.
▲ Presence of residual EDTA, e.g. when using medium containing EDTA for T cell purification, will hamper T cell stimulation. Ensure extensive removal of EDTA (i.e. over 200-fold reduction) prior to
T cell stimulation with the T Cell TransAct.
T Cell TransAct™
human
Order no. 130-111-160
2.1 Sample preparation
When working with anticoagulated peripheral blood or buffy coat, peripheral blood mononuclear cells (PBMCs) should be isolated by density gradient centrifugation, for example, using Ficoll-Paque™.
▲ Note:To remove platelets after density gradient separation, resuspend cell pellet in buffer and centrifuge at 200×g for 10−15 minutes at 20 °C. Carefully aspirate supernatant. Repeat washing step.
For details refer to the protocols section at www.miltenyibiotec/ protocols.
For the isolation of purified T cells use, for example, the Pan T Cell Isolation Kit, human.
2.2 T cell activation and expansion
The protocol has been optimized for gentle and efficient activation and expansion of purified T cells and PBMCs by using a titer of 1:100.
Purified T cells should be activated at an optimal surface density of 1×10⁶ cells per cm2 (table 1) and PBMCs with up to 2×10⁶ per cm2.
96 well0.31 cm²0.2 mL0.3×10⁶ 2 µL
48 well 1 cm² 1 mL1×10⁶10 µL
24 well 2 cm² 2 mL2×10⁶20 µL
12 well 4 cm² 4 mL4×10⁶40 µL
6 well10 cm² 5 mL5×10⁶50 µL
Table 1: Optimal surface densitiy when working with purified T cells. Volumes given below are for the stimulation in a 48-well plate of up to 1×10⁶ purified T cells or up to 2×10⁶ PBMCs in a total volume of 990 µL TexMACS™ Medium supplemented with 20 IU/mL Human IL-2 or 155 U/mL Human IL-7 and 290 U/mL Human IL-15. When working with fewer than 106 cells, use the same volumes as indicated in table 1. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly.
Activation in a 48-well plate
1. Determine cell number.
2. Resuspend cells in 990 µL supplemented TexMACS Medium.
3. Add 10 µL of the T Cell TransAct™.
4. Incubate at 37 °C, 5% CO₂ for up to 3 days.
▲ Note:Inspect culture daily, and add fresh medium if required.
5. Remove residual reagent 2–3 days after initial activation by
either replacing 900 µl of supernatant with fresh supplemented TexMACS Medium or by centrifugation at 300×g for
10 minutes and aspirate supernatant completely.
6. Add 1 mL fresh supplemented TexMACS Medium and incubate
at 37 °C, 5% CO₂.Expansion
1. Split cell suspension every 2 days into two equal parts and add
fresh supplemented TexMACS Medium.
2. Incubate at 37° C, 5% CO₂.
▲ Note:For optimal expansion of T cells a daily inspection of culture is
required. It might be necessary to split culture more or less frequently than
every day.
3. At day 14 proceed to downstream application, e.g., analysis of
cells.
4. (Optional) T cells can be further expanded by reapplying
T Cell TransAct to the culture. However, for restimulation it is
recommended to use a titer of 1:500.
2.3 Immunofluorescent staining
▲ Work fast, keep cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.
▲ It is recommended to stain 10⁶ cells per sample. When working with up to 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 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
▲ The recommended incubation temperature is 2–8 °C. Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice may require increased incubation times.
▲Upon stimulation, expression of CD3 will be transiently downregulated. Thus, the staining of CD3 on the cell surface of activated cells might be affected.
1. Determine cell number.
2. Wash cells by adding 1–2 mL of buffer per 10⁶ cells and
centrifuge at 300×g for 10 minutes. Aspirate supernatant
completely.
3. Add each staining antibody, e.g., CD4-VioBlue®,
CD8-VioGreen™, CD25-PE, and CD69-APC according to
manufacturer’s recommendations.
4. Mix well and incubate for 10 minutes in the dark in the
refrigerator (2–8 °C).
5. Wash cells by adding 1–2 mL of buffer per 10⁶ cells and
centrifuge at 300×g for 10 minutes. Aspirate supernatant
completely.
6. Resuspend cell pellet in a suitable amount of buffer for analysis
by flow cytometry or fluorescence microscopy.
3. Examples of T cell activation and expansion
王水提金using the T Cell TransAct™
A) Example of a T cell activation
Human purified T cells were isolated using the Pan T Cell Isolation K it and activated for 48 hours usi
ng the T Cell TransAct™ (titer 1:100) in TexMACS ™ Medium supplemented with Human IL-2 (20 IU/mL). The negative control experiment was performed without adding the T Cell TransAct. Cells were fluorescently stained using CD25-PE and CD69-APC and analyzed by flow cytometry using the MACSQuant® Analyzer. CD4-VioBlue® was used for selection of T helper cells and CD8-VioGreen ™ was used for selection of cytotoxic T cells. Dead cells and debris were excluded from the
analysis based on scatter signals and propidium iodide fluorescence.Negative control CD4+ T cells
CD8+ T cells
10³-1
0110¹10²010³10²10¹CD69-APC -11
10³
-1
0110¹10²010³10²10¹CD69-APC
-11
T cells after activation ++10³-1
0110¹10²010³10²10¹CD69-APC -11
10³
-1
0110¹10²010³10²10¹CD69-APC
-11
B) Expansion of pan T cells after activation
Isolated Pan T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with the T Cell TransAct. T cells were cultured at a density of 1×10⁶ cells per cm2 in supplemented TexMACS Medium supplemented with Human IL-2 (20 IU/mL). Proliferation analysis was done by flow cytometry via the detection of the CFSE dilution 7 days after stimulation. Non-stimulated pan T cells act as negative control. Unstimulated
Stimulated
Refer to www.miltenyibiotec for all data sheets and protocols.
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
榄
香烯乳状注射液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. autoMACS, MACS, the MACS logo, MACSQuant, TexMACS, TransAct, VioBlue, and
VioGreen are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide.
Ficoll-Paque is a trademark of GE Healthcare companies.
Copyright © 2018 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.
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