HSC migration assays were performed using transwells. Briefly, 150,000 HSCs were suspended in 100μl of starvation media (IMDM containing 1% (vol/vol) BSA fract V) and was added to the upper chamber of the 5-μm-pore transwell insert (24-well plate format transwell, Corning, NY, USA). 0.5ml of starvation media with various concentrations (0–100ng/ml) of chemokine stromal derived factor-1 alpha (SDF-1α) was added to the bottom chamber. The transwell plates were incubated for 4 hours in a 37°C 5% CO2. The transwell inserts were carefully removed and the migrated cells in the bottom chamber were resuspended. Samples were obtained from the bottom chamber and stained with acridine orange and propidium iodide nuclear dyes (AO-PI dye, Nexcelom, MA, USA). The stained samples were enumerated using Nexcelom auto 2000 cell counter (Nexcelom, MA, USA). All the migration experiments were performed in triplicates. In the experiments involving inhibitor treatments (LYN/Src inh. – RK20449 (Selleck Inc. TX, USA), SHIP1 inh. – 3AC (EMD Millipore Inc. MA, USA) and SHP1 inh. – TPI-1 (Cayman Inc. MI, USA)), the cells were exposed to inhibitors for 1 hour in IMDM containing 1% BSA media prior to adding to the top chamber of the transwell. Migration assays with TF1 cells were performed as described previously(27 ).
Auto 2000 cell counter
Manufactured by Revvity
Sourced in United States
The Auto 2000 cell counter is a laboratory instrument designed to automatically count and analyze cells. It provides accurate cell counts and viability measurements for various cell types.
Automatically generated - may contain errors
2 protocols using auto 2000 cell counter
1
Chemokine-induced Hematopoietic Stem Cell Migration
2
Luminex Assay for Anti-HIV-1 gp140 IgG
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We adapted the Luminex assay developed by C. Fenwick and coll. to detect anti-HIV-1 gp140z Env-specific IgG [23 (link)]. The in-house produced HIV-1 Env Gp140z trimer was associated with MagPlex beads using the manufacture’s protocol (Bio-Rad, France). Activated beads were washed in PBS followed by the addition of 6.3 μg of protein antigen (4°C overnight under agitation). Beads were then washed with PBS, resuspended in blocking buffer and finally in 150μl of storage buffer. Beads were counted with an Auto-2000 cell counter (Nexcelom) and kept protected from light at 4°C. Luminex beads were diluted at 50,000 beads/mL in PBS and 50μl was added in a Bio-Plex Pro 96-well Flat Bottom Plates (Bio-Rad). Following two 0.05% tween PBS on a magnetic plate washer (Bio-Rad), 50 μl of individual serum samples diluted at 1/100 in PBS, were added. Binding was performed at RT for 30 min under agitation, before adding an anti-mouse IgG-PE secondary antibody (45min at 0.5 μg/mL, ThermoFisher). Beads resuspended in 80 μl of Sheath fluid (Bio-Rad) were agitated 5 min at 700 rpm on the plate shaker then read directly on a Bioplex-200 plate reader (Bio-Rad) with 50μl of acquisition volume and DD gate 5,000–25,000 settings. Median fluorescence intensities (MFI) were exported using Bioplex Manager 6.1 software.
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