The authors thank Prof. Ruud Delwel and Prof. Ivo Touw for providing us with the 32D/G-CSF-R cell line, and Prof. Daniel G. Tenen for providing us with the Bosc23 cell line. This work was supported by grants of the Grant Agency of the Czech Republic (GACR 15-03796S and GACR 17-02177S) to MA-J, support from the Institute of Molecular Genetics of the Czech Academy of Sciences (RVO 68378050) to MA-J, a GA UK fellowship (project No. 341015) from Charles University in Prague to MK, and a GA UK fellowship (project No. 1278217) from Charles University in Prague to PD.

NOTE: Steps describing expansion, differentiation, and transduction of 32D/G-CSF-R cells are presented below.
1. Preparation
<ol>
	<li>Media preparation
	<ol>
		<li>Prepare 250 mL of culture medium: RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 10% heat inactivated FBS (fetal bovine serum) and murine IL-3 (10 ng/mL).
		<ol>
			<li>Alternatively, use home-made IL-3. To produce home-made IL-3, transduce HEK293 cells with IL-3 expressing vector and collect IL-3 containing supernatant22. 
			NOTE: Antibiotics, such as penicillin G (100 IU/mL), streptomycin (100 &#181;g/mL), and gentamicin (40 &#181;g/mL), can be used for cell culturing at any step of the protocol unless otherwise stated.</li>
		</ol>
		</li>
		<li>Prepare 50 mL of differentiation medium: RPMI 1640 medium supplemented with 10% FBS, and human G-CSF (100 ng/mL). 
		NOTE: (Important) Not all serum batches support differentiation of 32D/G-CSF-R cells. Prior to experiment start test different batches of serum. It is recommended to test at least 4 different batches and select the optimal based on the ability of the 32D/G-CSF-R cells to differentiate. See 32D/G-CSF-R differentiation protocol below.</li>
		<li>Prepare 10 mL of freezing medium: RPMI 1640 medium supplemented with 40% FBS, and 10% DMSO (dimethyl sulfoxide).</li>
	</ol>
	</li>
	<li>Production of retrovirus
	<ol>
		<li>Plate a single cell suspension of Bosc23 cells (6 &#215; 106) in a 16 cm Petri dish and cultivate in 18 mL of DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium) containing 10% FBS until the confluency of the culture reaches 80 % (24 h). 
		NOTE: Cells should grow in a monolayer and not form clumps in culture. Cell counting can be performed using different methods (valid for the rest of the protocols presented here). In case of low number of samples, manual counting under the microscope using B&#252;rker chamber is recommended. In this case, use Trypan blue for exclusion of dead cells. Mix cells 1:1 with 0.4% Trypan Blue in PBS. To perform counting of large number of samples, an automatic cell counter can be employed.</li>
		<li>Combine 40 &#181;g of retroviral construct (such as MSCV), 20 &#181;g of pCL-Eco (packaging vector)23, 80 &#181;l of PEI (polyethylenimine), and 2 mL of reduced-Serum medium (e.g. Opti-MEM) medium (without antibiotics). Incubate this mixture for 20 min at room temperature (RT).</li>
		<li>Carefully replace Bosc23 cells medium with 16 mL DMEM supplemented with 2% FBS. Pre-warm the medium to 37 &#176;C before use. 
		NOTE: Do not use antibiotics during transfection, since it may reduce transfection efficiency.</li>
		<li>Add the mixture prepared in 1.2.2. dropwise and carefully to the Bosc23 culture, and incubate for 4 h at 37 &#176;C. Limit incubation to less than 6 h due to PEI toxicity.</li>
		<li>After incubation, change Bosc23 medium to 18 mL pre-warmed DMEM containing 10% FBS, and cultivate cells for 48 h at 37 &#176;C. Place dishes in a biosafety level 2 laboratory, keep here from this step on, and follow standard safety procedures.</li>
		<li>Collect Bosc23 medium (containing ecotropic retroviral particles) using a 25-mL serological pipette into a 50-mL conical tube, and store at 4 &#176;C. 
		NOTE: (Important) Transfection efficiency should reach 90% to produce a high titer virus.</li>
		<li>Add 18 mL pre-warmed DMEM 10% FBS to Bosc23 cells and cultivate for 24 h.</li>
		<li>Repeat step 1.2.6. 
		NOTE: Retroviral supernatants from 1.2.6 and 1.2.8 can be pooled once they reach the same temperature (4 &#176;C) to preserve viral integrity.</li>
		<li>To avoid Bosc23 contamination in viral supernatants, spin collected virus at 1500 &#215; g for 10 min at 4 &#176;C. Aliquot virus, snap freeze using dry-ice or liquid nitrogen, and store at -80 &#176;C. However, note that freshly prepared virus is of higher quality in terms of infection efficiency than frozen stocks. 
		NOTE: (Important) Avoid repetitive freezing/thawing of virus, since it leads to viral degradation.</li>
	</ol>
	</li>
	<li>Production of lentivirus
	<ol>
		<li>Plate a single cell suspension of HEK293T (Human embryonic kidney 293T) cells (6 &#215; 106) in a 16-cm Petri dish and cultivate in 18 mL of DMEM containing 10% FBS until the confluency of the culture reaches 80 % (24 h). 
		NOTE: Cells should grow in a monolayer and not form clumps in culture.</li>
		<li>Combine 15 &#181;g of lentiviral construct (such as pGhU6), packaging plasmids pCMVdR8.74 (encoding for gag/pol, 12 &#181;g) and pMD2.VSVG (encoding for VSV-G, 1.4 &#181;g), 85.2 &#181;L PEI, and 2 mL of reduced-serum medium (without antibiotics). Incubate this mixture for 20 min at RT.</li>
		<li>Carefully replace HEK293T medium with 16 mL of DMEM supplemented with 2% FBS. Pre-warm the medium to 37 &#176;C before use. Do not use antibiotics during transfection, since it may reduce transfection efficiency.</li>
		<li>Carefully drop the mixture prepared in 1.3.2 to the HEK293T cell culture and incubate for 4 h at 37 &#176;C. Limit incubation duration to within 6 h to prevent PEI toxicity.</li>
		<li>Change medium to 18 mL pre-warmed DMEM 10% FBS and cultivate cells for 48 h at 37 &#176;C. Place dishes in a biosafety level 2 laboratory, keep here from this step on, and follow standard safety procedures.</li>
		<li>Collect HEK293T medium (containing amphotropic lentiviral particles) using a 25-mL serological pipette into a 50 mL conical tube and store it at 4 &#176;C. 
		NOTE: (Important) Transfection efficiency should reach 90% to produce a high titer virus.</li>
		<li>Carefully add 18 mL of pre-warmed DMEM 10% FBS to the HEK293T cells. Cultivate for 24 h at 37 &#176;C.</li>
		<li>Repeat step 1.3.6. 
		NOTE: Lentiviral supernatants from 1.3.6 and 1.3.8 can be pooled once they reach the same temperature (4 &#176;C) to preserve viral integrity.</li>
		<li>To avoid contamination of viral supernatant with HEK293T cells, spin collected virus at 1500 &#215; g for 10 min at 4 &#176;C. Aliquot virus, snap freeze using dry-ice or liquid nitrogen, and store it at -80 &#176;C. However, notice that freshly prepared virus is of higher quality in terms of infection efficiency than frozen stocks. 
		NOTE: (Important) Avoid repetitive freezing/thawing of virus, since it leads to viral degradation.</li>
	</ol>
	</li>
	<li>Titration of virus containing a GFP reporter
	<ol>
		<li>Seed 1 &#215; 105 NIH/3T3 cells in 300 &#181;L of DMEM 10% FBS per well into a 24-well plate. 7 wells will be employed to titer one virus.</li>
		<li>Thaw virus at 37 &#176;C and add 1, 5, 10, 50, 100, and 500 &#181;L virus to NIH/3T3 cultures. Do not add any virus into negative control well. Cultivate cells in the presence of the virus for 48 h at 37 &#176;C.</li>
		<li>Harvest NIH/3T3 cells using 30 &#181;L of trypsin/EDTA (ethylenediaminetetraacetic acid) (0.25% Trypsin, 0.01% EDTA in PBS) and place cells in 250 &#181;L PBS in a FACS (fluorescence activated cell sorting) tube.</li>
		<li>Determine the frequency of GFP+ cells by flow cytometry in each sample24. Exclude dead cells by addition of a viability dye, such as Hoechst 33258.</li>
		<li>Calculate the total numbers of GFP+ cells (based on the number of plated cells and percentage of GFP+ cells), and plot them against the volume of virus used for infection. 
		NOTE: (Important) Efficiency of infection reaches a plateau when large viral doses are applied. Therefore, it is important to estimate the titer based on viral concentrations at which infection efficiency occurs in a linear range (example shown in Table 1). The number of GFP+ cells indicates the number of functional viral particles (TU - transforming units) in a given virus volume. Recalculate the number of TU per mL.</li>
	</ol>
	</li>
	<li>Titration of virus containing a puromycin reporter
	<ol>
		<li>Seed 5 &#215; 104 NIH/3T3 cells in 3 mL of DMEM 10% FBS per well in a 6-well plate; employ 6 wells to titer one virus. Culture overnight at 37 &#176;C.</li>
		<li>Next day dilute virus as indicated in Table 2. To dilute virus, use pre-warmed DMEM 10% FBS. Do not vortex.</li>
		<li>Remove culture medium from NIH/3T3 cells and add 1 mL of diluted virus.</li>
		<li>Incubate overnight at 37 &#176;C.</li>
		<li>Gently replace the virus-containing medium with 2 mL of pre-warmed DMEM 10% FBS, and incubate overnight at 37 &#176;C.</li>
		<li>Gently replace NIH/3T3 medium with 2 mL of pre-warmed DMEM 10% FBS containing puromycin (2 &#181;g/mL).</li>
		<li>Culture at 37 &#176;C and carefully refresh medium with puromycin every 3 days or when medium turns yellow.</li>
		<li>At 10-12 days after infection, aspirate medium from each well and wash gently with PBS.</li>
		<li>Stain with 1 mL of crystal violet solution (0.5% crystal violet in 20% ethanol and dH2O), 2 min at RT, and wash carefully with PBS twice.</li>
		<li>Count blue colonies present in each well under a microscope (magnification X4). No colonies should be observed in the negative control well.</li>
		<li>Calculate the total numbers of TU/mL considering the dilution factor.</li>
	</ol>
	</li>
</ol>
2. Expansion and maintenance of 32D/G-CSF-R cells
<ol>
	<li>If 32D/G-CSF-R cells are frozen, take an aliquot and thaw cells in a 37 &#176;C water bath for 1 min and pour cells from the vial directly into 10 mL of pre-warmed RPMI 1640 medium supplemented with 10% FBS in a 15-mL tube. Invert the tube 3 times and spin the cells down at 400 &#215; g. Remove supernatant and resuspend cells in IL-3 containing culture medium at a concentration of 0.3 &#215; 106 cells/mL.</li>
	<li>Optimal cell growth concentration is 0.25-0.5 &#215; 106 cells/mL. Split cells every 2 days bringing them to a concentration of 0.1-0.2 &#215; 106 cells/mL. 
	NOTE: (Important) 32D/G-CSF-R cells may partially lose their ability to differentiate if grown to concentrations higher than 1 &#215; 106 cells/mL. Therefore, it is very important to split 32D/G-CSF-R cells on time.</li>
	<li>As needed, freeze and store cell aliquots for long periods of time at -80 &#176;C or in liquid nitrogen. Spin down 3 &#215; 106 cells, remove supernatant, and resuspend in 1 mL of freezing medium. Place tube in a freezing container at -80 &#176;C. For long term storage, reposition cells to liquid nitrogen.</li>
</ol>
3. Transduction of 32D/G-CSF-R cells
<ol>
	<li>Plate 32D/G-CSF-R cells into 6-well plate at a concentration of 0.3 &#215; 106 cells/mL.</li>
	<li>Add appropriate amount of virus to reach a MOI (multiplicity of infection) between 10 and 40. 
	NOTE: Higher MOI will result in increased percentage of GFP+ cells as well as higher levels of expression of the gene of interest. Example: To infect 0.3 &#215; 106 cells with a MOI of 10; 3 &#215; 106 TU will need to be added to the 32D/G-CSF-R cells.</li>
	<li>Add polybrene to final concentration 8 &#181;g/mL and incubate for 6 h at 37 &#176;C. Alternatively, perform a spin-inoculation procedure: centrifuge at 1200 &#215; g for 90 min at 30 &#176;C in a culturing plate using a swing-bucket rotor, and incubate for 3 h at 37 &#176;C.</li>
	<li>Collect cells, transfer to a 15-mL tube, and spin at 450 &#215; g for 5 min (4 &#176;C).</li>
	<li>Discard the supernatant, resuspend pelleted cells in 6 mL of culture medium, place in a T25 cell culture flask, and culture at 37 &#176;C.</li>
	<li>48 h later, harvest cells and spin them at 450 &#215; g for 5 min (4 &#176;C). Discard the supernatant.</li>
	<li>In case of a GFP reporter: place cells in 500 &#181;L of PBS and sort GFP+ cells using a flow cytometry sorter. In case of a puromycin reporter containing vector: place cells in culture medium containing 2 &#181;g/mL of puromycin.</li>
	<li>Expand cells as needed for further analysis and experiments. 
	NOTE: (Optional) Transduced cells can be frozen in freezing media in a freezing container at -80 &#176;C, and further stored in liquid nitrogen.</li>
</ol>
4. 32D/G-CSF-R cell proliferation assay
<ol>
	<li>To assess proliferation rate place 0.2 &#215; 106 cells in 1 mL of culture medium and incubate overnight at 37 &#176;C.</li>
	<li>Next day count number of cells and dilute them back to a concentration of 0.2 &#215; 106 cells/mL using culture medium. Incubate overnight at 37 &#176;C. Keep records of cell concentrations and dilutions applied to the daily cultures using Table 3.</li>
	<li>Repeat step 4.2 for as many days as proliferation needs to be assessed. 
	NOTE: The length of the proliferation assays will depend on the effect of the gene of interest. In case of a strong phenotype, 8-9 days might be sufficient to observe a significant effect (See Figure 3A). However, in case of a mild phenotype, longer periods of time might be required.</li>
	<li>Assess cell growth by making a proliferation curve as indicated in Table 3.</li>
</ol>
5. 32D/G-CSF-R cell differentiation assay
<ol>
	<li>Wash 0.2 &#215; 106 32D/G-CSF-R cells twice with RPMI medium without cytokines. 
	NOTE: The washing steps before addition of G-CSF into the culture are important, since presence of residual IL-3 may inhibit differentiation.</li>
	<li>Place cells in 1 mL differentiation medium (containing 100 ng/mL G-CSF) in a 24 well/plate, resulting in a cell concentration of 0.2 &#215; 106 cells/mL (Day 0). Culture overnight at 37 &#176;C.</li>
	<li>Count number of cells/mL as indicated above (step 1.2.1).</li>
	<li>Cytospin 2-5 &#215; 104 cells on a glass slide (76 mm X 26 mm) using a centrifuge with necessary adaptors at 20 &#215; g.</li>
	<li>Refresh differentiation medium and plate cells at a concentration of 0.2 &#215; 105 cells/mL on a 24 well/plate (Day 1). Discard the old plate, and proceed next day with step 5.6 with the new plate.</li>
	<li>On days 2 to 7, repeat steps 5.3 to 5.5. Assess cell proliferation in the presence of G-CSF as described in step 4. 
	NOTE: (Important) During differentiation, cell proliferation slows down, therefore after 3 to 4 days in the presence of G-CSF it is adequate to culture cells at higher concentrations.</li>
	<li>Assess differentiation state of 32D/G-CSF-R cells based on cell morphology.
	<ol>
		<li>Fix cells from step 5.4 in methanol for 5 min at RT and leave slides to air-dry. 
		NOTE: Slides from Day 0 to 7 can be stored at RT, and fixed simultaneously. Similarly, they can also be kept at RT for longer periods after fixation.</li>
		<li>Stain cells using May-Gr&#252;nwald Giemsa staining following manufacturer&#39;s protocol.</li>
		<li>Visualize stained cells using a microscope and magnification X40.</li>
		<li>Quantify number of blasts, intermediately differentiated cells, and mature granulocytes using illustrative pictures on Figure 1 and description in Table 4.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The choice of an experimental model is one of the main issues in research. Though primary animal and human cells are believed to produce the most biologically relevant data, these models may involve ethical concerns and are often associated with expensive and/or sophisticated isolation/culturing procedures. Primary cells are limited in numbers and it is hard to genetically manipulate them. In addition, primary cells represent a heterogeneous population composed of various cell types that may complicate data interpretatio...

The hematopoietic stem and progenitor population supplies the organism with a large range of mature cells, including cells from the myeloid lineage (neutrophils, eosinophils, basophils, and monocytes). The process that drives the production of myeloid cells from hematopoietic stem cells is known as myelopoiesis, and adequate production of mature myeloid cells in response to changing demands is a prerequisite for proper coping of the organism with stress conditions, such as infections and blood loss. Insufficient production of mature myeloid cells may lead to inability to eliminate pathogens, reduced blood coagulation, and other life-threatening conditions1,2. In addition, alterations in myeloid lineage development may also be associated with hematological malignancies, such as acute myeloid leukemia (AML)3. Alterations in myelopoiesis may occur due to various reasons, such as defects in cell surface receptors4, altered expressions of transcription factors5, impaired signaling pathways6, mutations resulting in formation/activation of oncogenes7, or inactivation of tumor suppressor genes8. 
Various methods have been developed to study myeloid development, and assess the effect of specific genetic alterations in this process. Common approaches used to study myelopoiesis involve primary cells and transgenic mice. Though these models allow acquisition of biologically relevant data, they have certain limitations. The use of primary cells encounters a limited number of cells and a restricted period of culture, narrowing the possibilities to alter gene expression and subsequent biological or biochemical analysis. Transgenic mice are costly and require a reasonable degree of biological justification. In addition, working with in vivo models adds a degree of complexity into understanding the role of a gene of interest in a given process. Therefore, alternative approaches to circumvent these limitations are needed. Cell lines have indisputable advantages: (1) they possess unlimited proliferation capacity that allows generating enough material for biochemical and biological studies, (2) they are susceptible to genetic manipulations (knockdown, knockout, overexpression), (3) the cost is relatively low, and (4) they allow a degree of biological simplification required in certain experimental approaches. 
The parental IL-3 (Interleukin-3) dependent 32D cell line was established in 1983 by Greenberger and colleagues by infection of bone marrow cells from C3H/HeJ mice with Friend murine leukemia virus9. Several 32D clones were described in literature: cl-239, cl-310, and cl-1011. The 32D cl-3 cells were shown to proliferate in IL-3 and undergo neutrophilic differentiation upon treatment with granulocyte-colony stimulation factor (G-CSF)10. On the contrary, 32D cl-10 cells, while being IL-3 dependent, originally were not differentiating in response to G-CSF treatment. In 1995 the group of Dr. Ivo Touw retrovirally transduced 32D cl-10 cells with wild type and mutant forms of G-CSF receptor (G-CSF-R), in order to identify functionally important regions of this receptor11. This study resulted in generation of the 32D/G-CSF-R cells, which are similarly dependent on IL-3, but within 6 to 10 days after replacement of IL-3 with G-CSF, cells stop to proliferate and irreversibly differentiate into mature neutrophils. These properties make 32D cl-3 and 32D/G-CSF-R cells simplified models of murine neutrophilic differentiation that can be modulated by two well-defined growth and differentiation factors - IL-3 and G-CSF. During the last decades multiple groups have used 32D/G-CSF-R cells to study the role of particular genes in proliferation and differentiation of myeloid cells in culture12,13,14,15,16, and to study G-CSF signaling17,18. Importantly, the results obtained using this cell line correlated with data obtained with primary cells and transgenic mice16,19,20,21. Consequently, we believe that 32D/G-CSF-R cells, being a widely used and well-established model, represent a valuable system to study myeloid differentiation which can be used in parallel with other approaches addressing this question. 
Here, detailed protocols describing handling of the 32D/G-CSF-R cell line, which cover expansion, differentiation, and assessment of proliferation and differentiation of these cells is presented. Detailed information for genetic modification of 32D/G-CSF-R cells, either by retroviral or lentiviral transduction, as well as protocols for virus titration are provided. In addition, several representative results that demonstrate potential applications of 32D/G-CSF-R cells are provided.

<table><tbody><tr><td>RPMI 1640 powder medium</td><td>Merck, Kenilworth, NJ, USA</td><td>T 121-10</td><td>without NaHCO3, with L-glutamine</td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>15028</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>31985-047</td><td>L-Glutamine, Phenol Red</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>PAA Laboratories (GE Healthcare,Chicago, IL, USA)</td><td>MT35011CV</td><td>For differentiation of 32D/G-CSF-R cells</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>10270</td><td>Used for culturing HEK293T, NIH3T3, BOSC23 cells</td></tr><tr><td>Penicillin</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>P3032</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>S9137</td><td>Streptomycin sulfate salt powder</td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>G1914</td><td></td></tr><tr><td>murine IL-3</td><td>PeproTech, Rocky Hill, NJ, USA</td><td>213-13</td><td></td></tr><tr><td>human G-CSF</td><td>PeproTech, Rocky Hill, NJ, USA</td><td>300-23</td><td></td></tr><tr><td>Polyethylenimine</td><td>Polyscience, Warrington, PA, USA</td><td>23966</td><td>Linear, MW 25,000 (PEI 25000)</td></tr><tr><td>Polybrene</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>H9268</td><td></td></tr><tr><td>Trypsin</td><td>VWR Chemicals, Radnor, PA, USA</td><td>0458</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>E5134</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>C0775</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>T6146</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>D2650</td><td></td></tr><tr><td>May-Gr&amp;uuml;nwald Giemsa</td><td>DiaPath, Martinengo, BG, Italy</td><td>10802</td><td></td></tr></tbody></table>

proliferation and differentiation of murine myeloid precursor 32d/g-csf-r cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Proliferation and differentiation of 32D/G-CSF-R cells
To assess proliferation of 32D/G-CSF-R cells under pro-proliferative and pro-differentiation conditions, 32D/G-CSF-R cells were cultured in media containing IL-3 and G-CSF, respectively. It was observed that cells cultured in IL-3 containing medium (10 ng/mL) divide approximately every 24 h (Figure 2A). Upon replacement of IL-3 with G-CSF (100 ng/m...

Watch this Scientific Journal Video about Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells at JoVE.com

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

proliferation-differentiation-murine-myeloid-precursor-32dg-csf-r

Research

JoVE Journal

Developmental Biology

9.9K Views.  Institute of Molecular Genetics of the ASCR. Hier worden gedetailleerde protocollen voor het kweken van de 32 quinquies/G-CSF-R cellijn van lymfkliertest myeloïde voorloper, uitvoeren van virale infecties en het uitvoeren van de proliferatie en differentiatie testen gepresenteerd. Deze cellijn is geschikt voor de studie van myeloïde cel ontwikkeling, en de rol van genen van belang in myeloïde celgroei en neutrofiele differentiatie.

Video: Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

Cancer Research

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Biology

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Immunology and Infection

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Medicine

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Source: Kozicky, L. et al., <a href="https://app.jove.com/v/56689">Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the activation of bone marrow-derived macrophages in response to bacterial lipopolysaccharide and intravenous immunoglobulin.

Source: Kozicky, L. et al., Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation. J. Vis. Exp. (2017)
In ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

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