Freshly isolated KLRG1bright NK cells were pre-activated with K562 target cells (effector : target ratio = 10:1) and 200 IU rhIL-2 (Miltenyi Biotech) for 48 hours. On day 2, cells were transduced with pHIV1-Siren lentiviral particles as previously described (32 (link)). On day 6 of culture, cells were transferred to a 96-well plate coated with an anti-KLRG1 mAb (BioLegend) or a IgG control Ab (BioLegend) both at 10 µg/ml and K562 target cells were added at an effector : target ratio = 10:1 and fresh IL-2 at 200 IU/ml. On day 9 of culture, apoptosis was assessed by Annexin V staining and proliferation by Ki67 staining. Telomerase reverse transcriptase (hTERT) expression was measured by intranuclear staining with the anti-TERT mAb (ab68781, Abcam). On day 9, NK cells were re-stimulated with K562 target cells at an effector : target ratio = 10:1 and fresh IL-2 at 200 IU/ml and cultured for a total of 12 days (for experimental design see Fig. S4A ).
Ab68781
Manufactured by Abcam
Sourced in United Kingdom
Ab68781 is a product from Abcam that functions as a laboratory equipment. The core function of this product is to provide a specific tool or instrument for use in scientific research and experiments. No further details on the intended use or application of this product are available.
Automatically generated - may contain errors
2 protocols using ab68781
1
Activating and Transducing KLRG1+ NK Cells
2
Endometrial Tissue Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Whole endometrial tissue lysate was prepared and homogenized in lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, and 0.25% sodium deoxycholate). Protein concentration was measured and adjusted by using the BCA Reagent kit (Applygen, Beijing, China). Samples were run on a 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in 5% nonfat dry milk in TBST (0.1% Tween 20 in TBS) for 1 h, membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-human telomerase (ab68781, Abcam, Cambridge, UK), ER alpha (sc-7207, Santa Cruz, CA, USA) or anti-human Gapdh (sc-25778, Santa Cruz). After three washes in TBST, membranes were incubated with secondary antibodies conjugated with horseradish perioxidase for 1 h at about 25°C. The signals were developed with the ECL Chemiluminescent kit (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). Films were scanned using a flat-bed scanner. The intensities of the bands representing ER alpha and TERT were evaluated using the Image J analysis software (Rawak Software, Inc., Germany).
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