For assays with ACH-2 cells,22 (link), 46 (link) 5.0 × 104 lentiviral vector-transduced DCs were cocultured with 2.0 × 105 ACH-2 cells in a 96 well plate or were plated at an equivalent ratio in the upper and lower chambers of a 0.4 μM transwell plate (Corning, Corning, NY). 1 μM PMA (Sigma, St. Louis, MO) was added to one well as a positive control. After 48 hours, the culture supernatant was harvested and infectious virus was plated on 1 × 104 TZM-bl reporter cells47 (link)-49 (link) in a 96 well plate. After two days, the plate was stained for β-galactosidase, and the spots were quantified with an ImmunoSpot S6 Micro Analyzer Elispot Reader (Cellular Technology, Ltd.). For assays with J-Lat cells,23 (link) the supernatant from cultures of 2.0 × 105 lentiviral vector-transduced DCs was collected 72 hours post-infection, serially diluted and applied to 2.0 × 105 J-Lat cells (full-length clone 10.6) in a 96 well dish. TNF-α (R&D Systems) was added to 1 ng/ml to one well as a positive control. After 24 hours, the GFP+ cells were quantified by flow cytometry. For select assays, 40 μl TNF-α (10 ng/ml) or 40 μl of culture supernatant from lentiviral vector-transduced DCs were pretreated with 2 mg/ml anti-TNF-α mAb for one hour and then added to the J-Lat cells.
Immunospot s6 micro analyzer elispot reader
Manufactured by Cellular Technology
The ImmunoSpot S6 Micro Analyzer is an Elispot reader designed for the quantification of secreted proteins from single cells. It is a compact and automated instrument that captures and analyzes Elispot images.
Automatically generated - may contain errors
Lab products found in correlation
0.4 μm transwell plate, by Corning (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Aec substrate, by BD (1 mentions)
Multiscreen hts plate, by Merck Group (1 mentions)
Immunocapture 6, by Cellular Technology (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Streptavidin hrp, by BD (1 mentions)
3 protocols using immunospot s6 micro analyzer elispot reader
1
HIV Latency Reactivation Assay
2
ELISPOT Assay for Virus-Specific T Cell Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Frozen PBMC were thawed in a water bath at 37°C, washed twice with thawing media (RPMI 1640 and 20% FBS), resuspended in culture media [RPMI 1640, 10% FBS, 1% antibiotic-antimycotic, and 55 μM 2-mercaptoethanol (Gibco)], and rested for 2 hours. Single cells isolated after lung digestion were not cryopreserved but used immediately. PBMC or lung cells were plated at 0.5 or 1 million live cells per well in 96 well MultiScreen HTS plates (Millipore, Billerica, MA) pre-coated with anti-IFN-γ (P2G10, BD Biosciences). The cells were then incubated at 37°C for 48 hours with 5 x 105 TCID50 of UV-inactivated TX98 or CA04 virus particles or virus-free UV-treated MDCK supernatant. Afterwards, the plates were developed using a biotin-conjugated anti-IFN-γ mAb (P2C11, BD Biosciences), streptavidin-HRP (BD Biosciences), and AEC substrate (BD Biosciences), according to manufacturer instructions. The number of spots in each well was read using an ImmunoSpot S6 Micro Analyzer ELISPOT reader with ImmunoCapture 6.4 software (Cellular Technology Ltd., Shaker Heights, OH). The data are presented as the number of spots per 106 PBMC or lung cells after subtracting the average number of spots in wells cultured with virus free MDCK supernatant.
3
HIV Latency Reactivation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For assays with ACH-2 cells,22 (link), 46 (link) 5.0 × 104 lentiviral vector-transduced DCs were cocultured with 2.0 × 105 ACH-2 cells in a 96 well plate or were plated at an equivalent ratio in the upper and lower chambers of a 0.4 μM transwell plate (Corning, Corning, NY). 1 μM PMA (Sigma, St. Louis, MO) was added to one well as a positive control. After 48 hours, the culture supernatant was harvested and infectious virus was plated on 1 × 104 TZM-bl reporter cells47 (link)-49 (link) in a 96 well plate. After two days, the plate was stained for β-galactosidase, and the spots were quantified with an ImmunoSpot S6 Micro Analyzer Elispot Reader (Cellular Technology, Ltd.). For assays with J-Lat cells,23 (link) the supernatant from cultures of 2.0 × 105 lentiviral vector-transduced DCs was collected 72 hours post-infection, serially diluted and applied to 2.0 × 105 J-Lat cells (full-length clone 10.6) in a 96 well dish. TNF-α (R&D Systems) was added to 1 ng/ml to one well as a positive control. After 24 hours, the GFP+ cells were quantified by flow cytometry. For select assays, 40 μl TNF-α (10 ng/ml) or 40 μl of culture supernatant from lentiviral vector-transduced DCs were pretreated with 2 mg/ml anti-TNF-α mAb for one hour and then added to the J-Lat cells.
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