Ex vivo PBMC cultures were undertaken as described previously [27 (link)]. In brief, 1.5x106 cells /ml suspended in RPMI 1640 (GIBCO NY, USA) with 10% pooled human AB serum, 2mM L-glutamine, 100 units of penicillin (Sigma Aldrich, MO, USA) and 100 μg streptomycin (Sigma Aldrich, MO, USA) were cultured in sterile flat bottom 24- well plates (Falcon, USA) as follows: cells were stimulated with and without 10 μg/ml of M. leprae sonicated antigen (MLSA) kindly provided by Dr. P J Brennan(Colorado State University, USA). Cultures were optimized for incubation in earlier studies [27 (link)] to 48 h at 37°C in humidified 5% CO2 + air. After incubation, harvested cells were washed as above and stored in RNA later (Sigma Aldrich, MO, USA) for gene expression studies or processed for flow cytometry analysis as given below.
Sterile flat bottom 24 well plates
Manufactured by BD
Sourced in United States
The Sterile flat bottom 24-well plates are laboratory equipment designed for cell culture applications. These plates provide a flat, sterile surface with 24 individual wells, each with a volume capacity suitable for various experimental setups. The core function of these plates is to serve as a container for culturing cells and performing related assays.
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Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Rnalater, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Ficoll, by Merck Group (1 mentions)
2 protocols using sterile flat bottom 24 well plates
1
Ex vivo PBMC Cultures for Leprosy Antigen Response
2
PBMC Isolation and Stimulation Protocol
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Three milliliter venous blood was collected from each CTB patients and HC. PBMC were isolated using Ficoll (Sigma) density-gradient method. Cell yields ranged from 1.3 to 1.5×106/mL, and cell viability ranged from 95% to 98% as estimated by 0.2% trypan blue staining (Sigma Aldrich). After isolation of PBMC, 2×106 cells/mL were cultured for 48 hours in sterile flat bottom 24-well plates (Falcon, Somerset, NJ, USA) with and without purified protein derivative (PPD) (2.5 µg/mL) antigens and 5 µg/mL phytohemagglutinin (Sigma) as positive control (Figures S1 and S2 ) in Roswell Park Memorial Institute-1640 medium +10% fetal bovine serum (Sigma Aldrich) for 48 hours in 5% CO2 at 37°C in incubator. After the stimulation, cells were analyzed by flow cytometry.
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