Bone marrow was carefully flushed from femurs as a single intact tissue and immediately hybridized with Alexa Fluor 488-labeled anti-GPIX (clone Xia.B4, Emfret), Alexa Fluor 647-labeled anti-CD31 (clone MEC13.3, BioLegend) and Hoechst 33258 (Roche) for 30 minutes before washing and overnight fixation in 3.7% buffered formaldehyde. Whole bone marrow tissue was mounted and visualized on a Leica DM4000 Confocal microscope (Wetzlar, Germany).
Hoechst 33258
Manufactured by Roche
Sourced in Switzerland, United States, Germany
Hoechst 33258 is a fluorescent dye used in various laboratory applications. It binds to the minor groove of DNA, exhibiting enhanced fluorescence upon binding. This property makes it useful for DNA detection and quantification in various assays.
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Lab products found in correlation
Axioplan 2 microscope, by Zeiss (2 mentions)
Axiocam hrc camera, by Zeiss (2 mentions)
Dm4000 confocal microscope, by Leica (1 mentions)
Lsrii flow cytometer, by FlowJo (1 mentions)
5 bromo 2 deoxyuridine brdu, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Bc 2800vet, by Mindray (1 mentions)
Chemray 800, by Rayto (1 mentions)
Fluorescence microscopy, by Leica (1 mentions)
Annexin 5 propidium iodide, by BD (1 mentions)
8 protocols using hoechst 33258
1
Bone Marrow Immunostaining Visualization
2
Hoechst 33258 Staining for Apoptosis
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Cell apoptosis was assessed using a minimum of 300 cells per treatment by Hoechst 33258 staining according to the manufacturer’s instructions. Cells were fixed with 4% paraformaldehyde for 20 min, then stained with Hoechst 33258 (3 μM) (Roche Diagnostics) in the dark for 5 min. Finally, cells were observed under the fluorescence microscope. In each group, five areas were randomly selected for analysis.
3
Cell Proliferation Assay with Idasanutlin and Venetoclax
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MV4-11 and MOLM-13 cells were treated with idasanutlin and venetoclax alone or in combination for 72 h (0.6–2000 nM). At the start of the final 24 h of incubation, 5-bromo-2-deoxyuridine (BrdU; Sigma) was added to cultures at a concentration of 80 μM. Culture medium was also supplemented with 80 μM deoxycytidine (Sigma) at this point to minimize disturbance to the nucleotide pathway. Prior to flow cytometric analysis, cells were washed twice in ice-cold DNA-staining buffer (100 mM Tris pH 7.4, 154 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1 % NP40, and 0.2 % bovine serum albumin) and incubated in DNA-staining buffer containing 10 U/mL RNase (Roche Diagnostics GmbH) and 1.5 μg/mL Hoechst 33258 for 15 min at 37 °C. Propidium iodide (PI) was added to a final concentration of 1.5 μg/mL, and cells were incubated on ice for 15 min. Fluorescence was analyzed on the LSRII flow cytometer, and data were analyzed using FlowJo software versions 7.6.5 and 10.0.7.
4
Quantifying Neuroblastoma/Glioma Cell Responses
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Neuroblastoma/glioma cells at 4 × 103 were seeded on glass coverslips in a 12-well plate for 24 h. Treatments with the indicated DMSO, CAPE, and RA concentrations were given for indicated time points followed by the fixation with pre-chilled absolute methanol at reverse transcriptase (RT) for 10 min. Fixed cells were subsequently permeablized with phosphate-buffered saline (PBS)-Triton-X-100 (0.2%) for 10 min followed by blocking with 2% bovine serum albumin (BSA) for 20 min. Indicated primary antibodies (please see the details of used antibodies in Supplementary Table 1 ) were incubated on RT for 1 h or at 4°C overnight. Cells were subsequently incubated with Alexa Fluor-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) and counterstained with Hoechst 33258 (Roche, Basel, Switzerland) as described earlier (Kalra et al., 2018 (link)). Immunofluorescence images were acquired under a Carl Zeiss Axioplan-2 microscope and captured with a Zeiss AxioCam HRc camera. The intensity of the acquired immunofluorescence images was quantitated by the ImageJ software that was further normalized with the respective controls and represented as % change over control.
5
In Vivo Blood Toxicity Evaluation
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All animal procedures were in agreement with the National Institutes of Health guidelines and approved by the ethics committee of the East China Normal University (ECNU). Specific pathogen–free female BALB/c nude mice of 4 weeks old with an average body weight of 20 g was purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China).
For in vivo blood toxicity assay, normal nude mice were divided into two groups (five mice in each group). PBS (150 μl) and F-P6a (150 nmol, 318 μg) were intravenously injected into the mice. The injections were repeated at the third and fifth day. The blood was collected 5 days after the last treatment, the liver function was tested by a biochemistry analyzer (Chemray-800, Rayto, China), and hematological parameters were measured by a hematology analyzer (BC-2800Vet, Mindray, China). The livers in treated mice were collected, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL). To determine the apoptosis level in liver tissues, a TUNEL assay was used. Briefly, the sections were incubated with TUNEL reaction mixture, proteinase K, and Hoechst 33258 according to the manufacturer’s protocol (Roche, Mannheim, Germany). The stained tissue sections were observed by a fluorescent microscope (Olympus, Japan).
For in vivo blood toxicity assay, normal nude mice were divided into two groups (five mice in each group). PBS (150 μl) and F-P6a (150 nmol, 318 μg) were intravenously injected into the mice. The injections were repeated at the third and fifth day. The blood was collected 5 days after the last treatment, the liver function was tested by a biochemistry analyzer (Chemray-800, Rayto, China), and hematological parameters were measured by a hematology analyzer (BC-2800Vet, Mindray, China). The livers in treated mice were collected, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL). To determine the apoptosis level in liver tissues, a TUNEL assay was used. Briefly, the sections were incubated with TUNEL reaction mixture, proteinase K, and Hoechst 33258 according to the manufacturer’s protocol (Roche, Mannheim, Germany). The stained tissue sections were observed by a fluorescent microscope (Olympus, Japan).
6
Immunofluorescence Analysis of Snol-A Treatment
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Cells were harvested and seeded (4 ×104 /well) on glass coverslips in a 12-well plate for 24 h. Treatments with indicated Snol-A concentrations were given for 48 h. The cells were fixed in pre-chilled methanol at room temperature for 5 min and then permeabilized with phosphate buffered saline (PBS)-Triton-X-100 (0.1%) for 10 min followed by blocking with 2% bovine serum albumin (BSA) for 20 min. Cells were probed with antibodies as indicated (β-catenin, hnRNP-K, Vimentin, Fibronectin, CARF, Cleaved PARP1/2, CDK2, p21WAF1 and Cyclin D1) at 4 °C overnight or at room temperature 1 h followed by incubation with Alexa Fluor-conjugated antibodies (Molecular Probes, USA) and then with Hoechst 33258 (Roche) for counterstaining. Immunofluorescence images were acquired on Carl Zeiss Axioplan-2 microscope equipped with Zeiss AxioCam HRc camera.
7
Apoptosis Confirmation by Fluorescence Microscopy
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To confirm the apoptosis induction found by comet assay, an aliquot of frozen lymphocytes obtained from the selected subjects (those resulted by comet assay with % apoptotic cells higher than 1% and identified from “Exp A” to “Exp J” for the exposed workers and “Contr A” for the control) was defrosted, centrifuged, and fixed with a solution of methanol/acetic acid (purity 99.90%, Carlo Erba, Italy) (3 : 1 v/v) for 30 min. Then, the cells were centrifuged, washed twice in PBS, incubated for 15 min at 37°C with 0.5 μg/ml Hoechst 33258 (Boehringer Mannheim, Germany) fluorescent dye, dropped onto slides, and covered with a coverslip (following Cavallo et al. [27 (link)] with minor modifications). Therefore, the cells were visualized for determination of nuclear chromatin morphology by fluorescence microscopy (Leica) at 1000x magnification. Apoptotic cells were recognized on the basis of nuclear condensation and/or fragmented chromatin. About 500 cells from each slide were examined for the presence of apoptotic features by an experienced observer, and % of total apoptotic cells was calculated (napoptotic on 500 examined cells × 100).
8
Apoptosis Analysis in HUVECs
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HUVECs were collected and fixed in methanol/acetone solution for 5 min and washed with PBS. Fixed cells were then stained with 0.1 ng/ml Hoechst 33258 (Boehringer Mannheim) for 10 min in the dark to counterstain the nuclei. The cells were observed and photographed under a Nikon fluorescence microscope. In some experiments, the Annexin V/propidium iodide (PI; BD Clontech) was used to quantify the number of apoptotic cells. Cells were washed twice with PBS and stained with Annexin V and PI for 20 min at room temperature. The level of apoptosis was determined by measuring the fluorescence of the cells by flow cytometry analysis.
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