BEL-A cells66 (link),67 (link), established in the lab of Deborah E. Daniels and Jan Frayne, were cultured in StemSpan™ SFEM (Stemcell Technologies) containing 50 ng/mL SCF, 3 U/mL EPO, 1 μM dexamethasone, and 1 µg/mL doxycycline. To induce erythroid differentiation, expanding cells were transferred to primary medium supplemented with 1 µg/mL doxycycline for 4 days, and for a further 4 days without doxycycline. Cells were cultured with supplementation of either 100 ng/mL FGF23 (R&D Systems) or vehicle control for 48 h before sample collection for analysis. Cell viability was determined by trypan blue exclusion test. For FACS, aliquots of 2 × 105 cells were incubated with band 3 primary antibody (BRIC71; IBGRL) in PBS containing 1% (w/v) BSA (Park Scientific Ltd) and 2 mg/mL glucose (PBS-AG), followed by APC-secondary antibody (BioLegend), or with conjugate antibodies (Annexin V-FITC or CD36-Vioblue; Miltenyi Biotech). Cells were analyzed on a BD LSR Fortessa flow cytometer. From day 6 of differentiation onwards cells were incubated with 5 μg/mL Hoechst 33342 nucleic acid stain (Merck) to distinguish the erythroblast and reticulocyte populations. Propidium iodide was used to exclude non-viable cells from analyses for band 3, CD36, and percentage reticulocyte measurements. Data were analyzed using FlowJo v10.6.1 (FlowJo LLC).
Hoechst 33342 nucleic acid stain
Manufactured by Merck Group
Hoechst 33342 is a fluorescent dye used to stain nucleic acids. It binds to the minor groove of DNA and exhibits blue fluorescence upon binding.
Automatically generated - may contain errors
Lab products found in correlation
Flowjo v 10, by FlowJo (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Fgf23, by R&D Systems (1 mentions)
Stemspan sfem, by STEMCELL (1 mentions)
Annexin 5 fitc, by Miltenyi Biotec (1 mentions)
Fitc conjugated cd 45, by Beckman Coulter (1 mentions)
Leica sp8 confocal microscope, by Leica camera (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
5 protocols using hoechst 33342 nucleic acid stain
1
FGF23 Modulates Erythroid Differentiation
2
Flow Cytometric Analysis of Erythroid Cells
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Aliquots of 2 × 105 cells were incubated with primary antibodies in PBS containing 1% (w/v) BSA (Park Scientific Ltd) and 2 mg⋅mL−1 glucose (PBS-AG), followed by secondary antibodies (along with conjugate antibodies when appropriate). Cells were analyzed on a BD LSR Fortessa flow cytometer. Propidium iodide was used to exclude non-viable cells from analyses. From mid-differentiation onward (day 6 for cell lines and day 9 for primary erythroid cultures) cells were incubated with 5 μg⋅mL−1 Hoechst 33342 nucleic acid stain (Merck) to distinguish the erythroblast and reticulocyte populations. Data were analyzed with FlowJo v.10.6.1 (FlowJo LLC).
3
Apoptosis Visualization in Cell Lines
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HCT116 and HT29 cells in their logarithmic growth phase were plated into 6-well plates and incubated for 48 h. The cells were then washed twice with PBS, incubated in the dark for 10 min with Hoechst 33342 nucleic acid stain (Sigma-Aldrich; Merck KGaA), and subsequently examined for their nuclear morphology under a fluorescence microscope (Olympus Corp.).
4
Immunophenotyping of PB-NHESC-C Cells
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Samples of PB-NHESC-Cs were fixed with 3.5% paraformaldehyde for 20 min, pre-blocked with 2% bovine serum albumin for 10 min at RT, and subsequently stained with fluorescein isothiocyanate (FITC)-conjugated CD45 (1:100, mouse monoclonal IgG; Beckman Coulter) for 30 min at RT in the dark. Hoechst 33342 nucleic acid stain (Sigma Aldrich) was added at 10 μg/mL to the cell suspensions for 20 min at RT in the dark. After washing, PB-NHESC-C was acquired using a laser scanning microscope (Leica SP8 confocal microscope, Leica) with FITC (emission 496–598 nm) and Hoechst (emission 415–470 nm) channels using a 60x objective.
5
Immunofluorescence Staining Protocol
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Cells were fixed with 4% paraformaldehyde (PFA), washed with phosphate buffered saline (PBS), incubated in blocking solution for 1 hour, and incubated with primary antibodies at 4°C overnight (see Supplemental Table 2
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