Dynabeads untouched mouse cd8 cells, by Thermo Fisher Scientific - Product details

1

PDAC Organoids Potentiate CD8+ T Cell Killing

Check if the same lab product or an alternative is used in the 5 most similar protocols

Mouse PDAC cells stably expressing OVA and carrying doxycycline (Dox)-inducible mTurquoise2-Atg4B^C74A were grown as organoids, treated with or without Dox (1 μg/mL) for 96 hrs. Organoids were dissociated into single cells, which were then incubated with either anti-H-2K^b-SIINFEKL antibody (clone 25-D1.16, BioXCell, BE0207) or isotype control (clone MOPC-21, BioXCell, BE0083) at 100 μg/mL for 30 min at 4 °C. Total splenocytes were harvested from OT-I mice and CD8⁺ T cells were enriched using Dynabeads® Untouched™ Mouse CD8 Cells (Invitrogen, 11417D) following manufacturer’s instructions. Isolated CD8⁺ T cells were labelled with 10 μM CFSE (BioLegend) for 10 min at RT in the dark, washed 3x with RPMI-1640 supplemented with 10% FBS. Ten thousand PDAC cells and forty thousand CD8⁺ cells were seeded in 96-well plates and cultured in 100 μL 50% DMEM and 50% RPMI-1640 supplemented with 10% FBS, 10 ng/mL recombinant murine IL-2 (Peprotech), 27.5 μM 2-Mercaptoehanol (Gibco), and 100 μg/mL of respective antibodies. After 48 hrs, CD8⁺ T cells were harvested and stained with anti-CD8a antibody (AF647, clone 53–6.7, BioLegend) and DAPI, and proliferation was analyzed by CFSE dilution using flow cytometry. After removal of CD8⁺ T cells, the viability of remaining PDAC cells were measured by CellTiter-Glo (Promega).

Yamamoto K., Venida A., Yano J., Biancur D.E., Kakiuchi M., Gupta S., Sohn A.S., Mukhopadhyay S., Lin E.Y., Parker S.J., Banh R.S., Paulo J.A., Wen K.W., Debnath J., Kim G.E., Mancias J.D., Fearon D.T., Perera R.M, & Kimmelman A.C. (2020). Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature, 581(7806), 100-105.

+ Open protocol

+ Expand

2

Isolation of CD8+ and CD4+ T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

CD8+ and CD4+ cells were isolated by negative selection (Dynabeads untouched mouse CD8+ cells or CD4+ cell kit, respectively; Invitrogen) from total splenocytes or total lymph node single cell suspensions, following manufacture's instruction. In brief, total splenocytes or LN cells after red cell lysis were adjusted to a concentration of 5×10⁷ cells in 0.5 ml isolation buffer and 0.1 ml FBS was added. Cells were incubated with 0.1 ml antibody mix for CD8+ or CD4+ cells for 20 min at 4°C. Cells were washed with isolation buffer, and then 1 ml Mouse Depletion Dynabeads was added to the cells. After 15 min incubation at room temperature, cells were washed and magnetic separation was performed. Total CD8+ or CD4+ cells were present in the supernatant. Enriched T-cell preparations were analyzed by flow cytometry using anti-CD3, anti-CD4 and anti-CD8 mAbs and > 90% cell purity for either CD8+ or CD4+ T-cell subpopulation was confirmed.

Manrique S.Z., Dominguez A.L., Mirza N., Spencer C.D., Bradley J.M., Finke J.H., Lee J.J., Pease L.R., Gendler S.J, & Cohen P.A. (2016). Definitive activation of endogenous antitumor immunity by repetitive cycles of cyclophosphamide with interspersed Toll-like receptor agonists. Oncotarget, 7(28), 42919-42942.

+ Open protocol

+ Expand

3

PDAC Organoids Potentiate CD8+ T Cell Killing

Check if the same lab product or an alternative is used in the 5 most similar protocols

Mouse PDAC cells stably expressing OVA and carrying doxycycline (Dox)-inducible mTurquoise2-Atg4B^C74A were grown as organoids, treated with or without Dox (1 μg/mL) for 96 hrs. Organoids were dissociated into single cells, which were then incubated with either anti-H-2K^b-SIINFEKL antibody (clone 25-D1.16, BioXCell, BE0207) or isotype control (clone MOPC-21, BioXCell, BE0083) at 100 μg/mL for 30 min at 4 °C. Total splenocytes were harvested from OT-I mice and CD8⁺ T cells were enriched using Dynabeads® Untouched™ Mouse CD8 Cells (Invitrogen, 11417D) following manufacturer’s instructions. Isolated CD8⁺ T cells were labelled with 10 μM CFSE (BioLegend) for 10 min at RT in the dark, washed 3x with RPMI-1640 supplemented with 10% FBS. Ten thousand PDAC cells and forty thousand CD8⁺ cells were seeded in 96-well plates and cultured in 100 μL 50% DMEM and 50% RPMI-1640 supplemented with 10% FBS, 10 ng/mL recombinant murine IL-2 (Peprotech), 27.5 μM 2-Mercaptoehanol (Gibco), and 100 μg/mL of respective antibodies. After 48 hrs, CD8⁺ T cells were harvested and stained with anti-CD8a antibody (AF647, clone 53–6.7, BioLegend) and DAPI, and proliferation was analyzed by CFSE dilution using flow cytometry. After removal of CD8⁺ T cells, the viability of remaining PDAC cells were measured by CellTiter-Glo (Promega).

Yamamoto K., Venida A., Yano J., Biancur D.E., Kakiuchi M., Gupta S., Sohn A.S., Mukhopadhyay S., Lin E.Y., Parker S.J., Banh R.S., Paulo J.A., Wen K.W., Debnath J., Kim G.E., Mancias J.D., Fearon D.T., Perera R.M, & Kimmelman A.C. (2020). Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature, 581(7806), 100-105.

+ Open protocol

+ Expand

4

CD8+ T Cell Proliferation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

CD8⁺ T cells were purified from WT and Arhgap45^−/− OT‐I mice by immunomagnetic negative selection using Dynabeads^® Untouched™ Mouse CD8 cells, (Invitrogen). Purified T cells were stimulated with irradiated H‐2 K^b‐ positive spleen cells isolated from T‐cell‐deficient mice and pulsed for 2 h with the N4 agonist OVA peptide or with PMA and ionomycin. After 48 h of culture, T‐cell proliferation was assessed with CellTiter‐Glo^® Luminescent Cell Viability Assay (Promega). The resulting luminescence, which is proportional to the ATP content of the culture, was measured with a Victor 2 luminometer (Wallac, Perkin Elmer Life Science).

He L., Valignat M., Zhang L., Gelard L., Zhang F., Le Guen V., Audebert S., Camoin L., Fossum E., Bogen B., Wang H., Henri S., Roncagalli R., Theodoly O., Liang Y., Malissen M, & Malissen B. (2021). ARHGAP45 controls naïve T‐ and B‐cell entry into lymph nodes and T‐cell progenitor thymus seeding. EMBO Reports, 22(4), e52196.

+ Open protocol

+ Expand

5

Transwell Assay for T and B Cell Migration

Check if the same lab product or an alternative is used in the 5 most similar protocols

Migration was studied with a Transwell tissue culture system (Corning) containing insert membrane with 5 μm pores. 0.5 × 10⁶ isolated CD4⁺ T cells (EasySep™ Mouse CD4⁺ T‐cell isolation Kit, STEMCELL™) or isolated CD8⁺ T cells (Dynabeads^® Untouched™ Mouse CD8 cells, Invitrogen) from WT, Arhgap^−/− or Arhgap45^∆T/∆T mice were added to the upper chamber and allowed to transmigrate for 2 h at 37°C in the presence or absence of 100 μg/ml of CCL19, CCL21, or CXCL12 (all from PeproTech^®) in the lower chamber. When specified, T cells were activated for 2 days with plate‐bound anti‐CD3 at 3 µg/ml (145‐2C11; Exbio) and soluble anti‐CD28 at 1 µg/ml (37‐51; Exbio), rested for 2 days in IL‐2 prior to be tested in Transwell migration assay. Cells that had migrated to the bottom chamber were counted using flow cytometry after adding 10 µl of cell counting beads (CountBright™, Invitrogen). Migration rate was measured as percentage of migrated cells relative to total input ([no. of migrating cells/no. of input cells] × 100). 0.5 × 10⁶ purified B cells (Dynabeads^® Untouched™ Mouse B cells, Invitrogen) were similarly analyzed for their ability to transmigrate for 4h in the presence or absence of 500 µg/ml of the specified chemokines in the lower chamber. When specified, insert membrane was coated with 10 µg/ml of recombinant mouse ICAM‐1 (R&D Systems) or PBS 1× for 4 h prior to the migration test.

He L., Valignat M., Zhang L., Gelard L., Zhang F., Le Guen V., Audebert S., Camoin L., Fossum E., Bogen B., Wang H., Henri S., Roncagalli R., Theodoly O., Liang Y., Malissen M, & Malissen B. (2021). ARHGAP45 controls naïve T‐ and B‐cell entry into lymph nodes and T‐cell progenitor thymus seeding. EMBO Reports, 22(4), e52196.

+ Open protocol

+ Expand

Dynabeads untouched mouse cd8 cells

Lab products found in correlation

Close spelling variants of this product

5 protocols using dynabeads untouched mouse cd8 cells

PDAC Organoids Potentiate CD8+ T Cell Killing

Isolation of CD8+ and CD4+ T Cells

PDAC Organoids Potentiate CD8+ T Cell Killing

CD8+ T Cell Proliferation Assay

Transwell Assay for T and B Cell Migration

About PubCompare

Ready to get started?