Two groups (n = 3) of rats were sacrificed to count erythroid cells in the bone marrow before rHuEPO treatment and after five doses of 450 IU/kg rHuEPO stimulation. Single-cell suspensions from bone marrow were collected from the rat femur and then counted with trypan blue staining by an automated cell counter (Invitrogen™ Countess™ 3, Thermo Fisher Scientific, Waltham, MA, United States). Bio-conjugated anti-rat erythroid cell HIS-49 and PE-conjugated anti-rat CD71 antibodies (BD Biosciences, San Jose, CA, United States) were used for identifying erythroid cells. Flow cytometry analysis was conducted using a BD LSR Fortessa Cell Analyzer (BD Biosciences, San Jose, CA, United States). Erythroid cells were stratified into three populations (early erythroblasts, late erythroblasts, reticulocytes) based on forward scatter and CD71 levels, as previously described (Yan et al., 2013 (link)). The gating strategy is shown in Figure 4A . The absolute number of subpopulation cells was calculated by percentage × the total number of living cells in one femur.
Invitrogen countess 3
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Invitrogen™ Countess™ 3 is a compact and automated cell counter that provides accurate cell counts and viability analysis. It utilizes advanced image-based technology to rapidly and reliably measure cell concentration and viability in a wide range of sample types.
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2 protocols using invitrogen countess 3
1
Quantifying Erythroid Cell Populations
2
CYP4F11 Knockdown Viability Assay
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The siCYP4F11 cells were seeded 24h post-transfection at the concentration of 5,000 cells/well in 96-well plates. The consequences on cell viability were determined 48 h posttransfection. The proliferation of the cells was detected by cell counting using the automated cell counter Invitrogen Countess3 (Thermo Fisher) and MTT Cell Proliferation Assay Kit (Abcam) following the protocol. The absorbance was measured at 590 nm using a microplate reader (BioTek). The data was normalized using GraphPad Prism to report cell viability [100%].
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