Hl 60 cell culture protocol pdf

The cellcycle kinetics, apoptosis and dna damage were assessed using apoptosis, dna damage and cell proliferation kit bd biosciences, san jose, ca, usa accordingly to the manufacturers protocol. Rouxmaintained embryonic chick cells in a saline culture. Although the cell lines doubling times were originally reported to be 55 to 60 h, once it is established, cell density doubles every 24 h in an actively growing culture, although doubling time is. The addition of antibiotics to media during transfection may result in cell death, and has not been tested for hl 60 cells. Hl, hl 60 human caucasian promyelocytic leukaemia the hl 60 cell line was derived from peripheral blood leukocytes obtained by leukopheresis of a 36yearold caucasian female with acute promyelocytic leukemia. Synergistic activity of parthenolide and cytarabine in. Ridley aj, schwartz ma, burridge k, firtel ra, ginsberg mh, borisy g, parsons jt, horwitz ar. Hl 60 cells spontaneously differentiate and differentiation can be stimulated by butyrate, hypoxanthine, phorbol myristic acid pma, tpa, dimethylsulfoxide dmso, 1% to 1. Under these conditions, hl 60 cultures can be maintained by diluting the cells with a fresh medium to a density of 10 5 viable cellsml when the cell density reaches 10 6 cellsml. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility.

Animal cell culture protocol aseptic technique and good cell culture practice to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Transfecting plasmid dna into hl60 cells using lipofectamine. Resuspend the cells in 20 ml rpmi containing 1% bsa. We have found that retinoic acidtreatment of hl 60 cells over a period of 68 days resulted in a progressive increase in the proportion of cells with mature neutrophil morphologies and was closely followed by an increase in the proportion of cells exhibiting the morphological characteristics of apoptosis, the nonpathological mode of cell death. Hl60 cell avalanche transfection reagent is a new class of unique chemical formulations specifically formulated and optimized for transfecting hl60 cells. Hl 60 is a nonadherent promyelocytic cell line derived by s. The tumorigenic hl 60 cell line was established using the peripheral blood tissue from a 36yearold female patient who suffered from acute promyelocytic leukemia apl. Discard supernatant, resuspend cells in 20 ml prewarmed complete cell culture medium and transfer cells into one t75 cell culture flask. Weve got everything else youll need to get started in your research of your hl1 cells. Hl60 cells were treated with ppm1d inhibitor sl176 7. Characterization of the continuous, differentiating myeloid cell line hl 60. Hl 60 cultures oct262009 hi, i am planning to use hl 60 cell cultures and i realized there are differences in culture conditions, for example medium rpmi in some labs has 5% fbs, in others it has 10% fbs. Hl 60 culture hematopoietic stem cells mar162007 hi everybody, i am looking for a protocol to for hl 60 cell ine culture. Hl60 cell growthconditioned medium is an effective.

Materials and methods cell culture and differentiation condition the hl 60 cells atcc, ccl240, rockville, md were grown in. Standardization of an opsonophagocytic assay for the. This data would be useful in optimizing the differentiation protocol of hl 60 cells for use in opka to s. Phenotypic and functional analysis of hl60 cells used in. I am culturing hl60 cells in life sciences rpmi, glutamax adding 10% fcs. Hl 60 cells were obtained from the riken gene bank tsukuba, japan and were maintained in an rpmi1640 medium supplemented with 10% fetal bovine serum fbs. Hl60 differentiation to neutrophilsmonocytes the open lab.

Peripheral blood leukocytes were obtained by leukopheresis from a 36yearold caucasian female with acute promyelocytic leukemia. Dna cell cycle analysis was performed by flow cytometry. Hl60 cells were seeded in 6well plates at a density of 2. Culture guides culturing biological materials can be challenging at times even for the expert culturist. Mix well to ensure sufficient separation of the cells. It was among the first longterm suspension cultures of human myeloid leukaemic cells to be established.

There is an immense body of literature on hl 60, therefore it was chosen for modeling. Thus, if 200,000 hl 60 target cells are used per well in a cytotoxicity assay, it will be. Inhibition of serthr phosphatase ppm1d induces neutrophil. Adjust the cell density of the suspension to 2 to 3 x 105 viable cellsml in the shipping medium. Alternatively, cultures can be established by centrifugation with subsequent resuspension at 1x10 5. Hl60 cells are predominantly a neutrophilic promyelocyte, used for laboratory research on how certain kinds of blood cells are formed. Hl60 cultures tissue and cell culture protocol online. Gently pellet the cells by centrifugation 35 minutes at 1,000 2,000rpm, 4 oc. Hl 60 cell preparation hl 60 is a nonadherent promyelocytic cell line derived by s. Hl60 proliferates continuously in suspension culture in nutrient and antibiotic chemicals. May 22, 2018 rsv inhibits cell proliferation and induces cell death in hl 60 cells. Monocytemacrophage like cell differentiation induced by. Wash cells once in pbs buffer gibco add 2ml of tryple solution gibco to the cells incubate cell for 2 minutes at 37c or until completely detached from the plate.

After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. Maintaining healthy cells is the key to experimental success and reproducible research results. Harvest 1 do not use cells that have been passed more than 25 times 2 remove cells from flasks according to protocol described above under subculturing 3 examine viability using trypan blue staining. The culture media used for cell cultures are generally quite complex, and culture condition widely varies for each cell type. Differentiation of hl 60 to neutrophil or monocytelike cells by vitamin a atra and vitamin d 1a,25oh2d3 seed cells at 2. This protocol assumes that the cells are being cultured in t150 flasks equipment equipment.

With over 85 years of expertise with cell and microbial cultures, atcc has acquired and developed a vast body of best practices to aid researchers at all levels of proficiency to maximize the return on their biomaterials investment. Rahul kumar vempati, school of life sciences, university of madras, guindy campus, chennai, india, email. Use of hl60 cell line to measure opsonic capacity of. Hl60 is a human caucasian promyelocytic leukemia cell line figure 7. A promyelocytic cell line derived from a patient with acute promyelocytic leukemia. Clone 15 hl60 was picked from soft agar being selected for the ability to differentiate to eosinophils after treatment with butyric acid. Materials and methods cells and culture conditions. For hl 60, lysis of 200,000 cells per well produces a strong a490 value of 0. The proprietary formulation of lipids and polymers ensures the highest possible transfection efficiencies and viabilities for hl60 cells. The hl 60 leukemia cell line is characterized by high levels of expression of cmyc due to gene amplification 84,85. Dgk expression was assessed through northern blot, end. Check which culture media and culture supplements the cell line you are using requires before starting cultures.

Intracellular flow cytometric detection of mpo in k562. Hl 60 cell lines may even be maintained on serumfree medium as originally described by breitman et al. But when subcultured from the first flask to new flasks cell solution. Spin cells down for 5 min at 200 x g at room temperature rt. Quickly transfer the ampoule to a 37c water bath until only one or two small ice crystals, if any, remain 12 minutes. These cells positively express the myc oncogene, and was derived by s. Hl60 is a suspension cell line with a lymphoblastic morphology. Cultures can be maintained by the addition of fresh medium or replacement of medium. Although the cells are capable of reverting to the parental phenotype, most passages maintain a high degree of eosinophil differentiation. Mammalian cell tissue culture techniques protocol abcam.

Hl 60 is a nci 60 cell line that is a widely used model for studying the mechanism of action of atra. Data are representative of three independent experiments. A model system for studying human myeloid cell differentiation. Clone 15 hl 60 was picked from soft agar being selected for the ability to differentiate to eosinophils after treatment with butyric acid. Cells of the tissue culture cell line hl 60 promyelocytic leukemia cells. Loeb demonstrated the survival of cells isolated from blood and connective tissue in serum and plasma. Under these conditions, hl 60 cultures can be maintained by diluting the cells with a fresh medium to a density of 10 5 viable cells ml when the cell density reaches 10 6 cells ml.

Molecular mechanisms of apoptosis induced by a novel. However, on the basis of a resistance of an hl 60 cell variant to both 1,25oh2d3 and pma, we can assume that these 2 agents may require a similar cellular event in the induction of a macrophagelike cell differentiation in the hl 60 cells. However, media generally include amino acids, vitamins, salts maintain osmotic pressure, glucose, a bicarbonate buffer system maintains a ph between 7. The clone 15 hl60 has been derived from hl 60 cells ecacc catalogue no. The day of transfection, count the cells to determine culture density. Alternatively, cultures can be established by centrifugation with subsequent resuspension at 1x10 5 viable cells ml. Protocol for hela cell culture and plasmid transfection av02hela cell culture 1. For optimum results, cells for cryopreservation should be in log phase of growth. All handling of the rnp reagents was performed in a class ii a2 biological safety cabinet to avoid contamination of the cell lines. Dna was isolated from the hl 60 cell culture using the qiaamp dna blood mini kit qiagen inc. The hl60 cells were maintained in rpmi1640 medium gibco supplemented with 10% calf serum and 2 nmoll glutaminein at 37c in a humidified atmosphere of 5% co 2 in air. Hl 60 dna dna from the hl 60 cell culture was extracted, isolated, and quantified by the professional services department of the american type culture collection a tcc, manassas, va. The cell culture flask, 1xt25, comes filled with cell culture medium. Using bioinformatics and in vitro experiments, we identified the microrna gene expression profile of hl 60 cells during atra induced granulocytic differentiation.

Incubate the culture, horizontally, at 37c in a 5% co2 in air. Hl60 differentiation to neutrophilsmonocytes the open. Resveratrol induces autophagydependent apoptosis in hl60. Clone, hl 60, hl60 human caucasian promyelocytic leukaemia the clone 15 hl 60 has been derived from hl 60 cells ecacc catalogue no. If the dilution method is chosen, the medium can be replaced by fresh growth medium. If you wish to use antibiotics during transfection, test your conditions thoroughly.

The human leukemia cell line hl60 as a cell culture model. Hl60 atcc ccl240 homo sapiens peripheral blood acute p. Hl 60 cells are passaged when the cells reach a density between 12 million cells ml in 25 cm2 cell culture flasks with 0. You may need to determine empirically the optimal cell number or spheroid size, apoptosis induction treatment and incubation time for the cell culture system. Transfection of hl 60 cells use this procedure to transfect plasmid dna into hl 60 cells in a 24well format for other formats, see scaling up or down transfections, below. Apr 25, 2011 differentiation of the acute myeloid leukemia aml cell line hl 60 can be induced by all transretinoic acid atra. Collins sj 1987 the hl 60 promyelocytic leukemia cell line. Leukemia cell line an overview sciencedirect topics. A perfusionculturing method with cell retention was found to be most effective for highdensity production of hl60 cells at a 21 as well as a 60 1 fermentor scale. The following is a general guideline for culturing of cell lines. The hl 60 cell line derived from a patient with promyelocytic leukemia may be grown in liquid suspension cell culture. Similar or standard freezing protocols may be substituted.

If seeding to plates, seed to basedensity of approximately 1x106 cells 25cm2 e. Protocol for hela cell culture and plasmid transfection av. Unless imaging, all cell work is performed under a biological safety cabinet sg303, the baker company. Although 80% of the cells will exhibit this differentiation pattern initially, reversion will occur during culture leading to a higher percentage reverting to neutrophilic differentiation. Claude bernardproposed that physiological systems of an organism can be maintained in aliving system after the death of an organism. Does anyone have a protocol for expanding hl60 from. This cell line exhibits myeloblastic cell morphology and a promyeloblast cell type. Eos hl 60 and hl 60 have been shown to be identical genetically by str. To give you confidence in the health of your cells every step of the way, weve highlighted the technologies and products within cell biology that are critical to maintaining optimal cell health.

To give you confidence in the health of your cells every step of the way, weve highlighted the technologies and products within cell biology that are critical to maintaining optimal cell. Hl60 cells were cultured in rpmi 1640 standard medium with 2 mmoll lglutamine supplemented with 10% heatinactivated fetal calf serum, 100 uml penicillin and 100. Every step of the way, a wide range of cell health products maintaining healthy cells is the key to experimental success and reproducible research results. They also exhibit phagocytic activity and responsiveness to chemotactic stimuli. All amounts and volumes are given on a per well basis. General details of cell culturing and subculturing.

If seeding to plates, seed to basedensity of approximately 1x106 cells25cm2 e. Human promyelocytic leukaemic cells established from hl60 cells by subcloning in methylcellulose. A lichen substance as an antiproliferative compound. Hl60 cells are passaged when the cells reach a density between 12 million cellsml in 25 cm2 cell culture flasks with 0.

An effective model of the retinoic acid induced hl60. The cells are now ready to be seeded and should be sufficient for two 96well cell culture plates at a density of 1 x 105 5 x 105 cells well. Graded increase in probability of eosinophilic differentiation of hl 60 promyelocytic leukemia cells induced by culture under alkaline conditions. In addition, k562 cells incubated in hl 60 gcm demonstrated a significant increase in the surface presentation of cd11b at days 1 and 2, as detected by flow cytometry 6% reactive cells in the control culture and 42% in the hl 60 gcm culture. Hl 60 cells require simple maintenance in vitro and grow as single cell suspension cultures without the tendency to clump or adhere to plastic or glass. The hl60 cell line is a human leukemia cell line that has been used for laboratory research on blood cell formation and physiology. Hl60, acute promyelocytic leukemia atcc ucsc genome browser. Materials and methods cell culture and treatment with tpa hl 60 cells were grown in rpmi1640 medium supplemented correspondence to. Optimization of culture conditions for high cell density proliferation of. Immediately dilute and gently mix each 1ml of cells with 10ml of complete cell culture medium. Comparison of the complete mtdna genome sequences of. It is known that around 10% of hl60 cells spontaneously differentiate gallagher et al, 1979. It binds 125iiodoinsulin in a highly specific, rapid, reversible.

Nov 04, 2019 suggest you get new hl 60 cells from a reliable source, follow a protocol which removes the dmso as much as possible on the first days of culture, and then try again. To investigate the action of rsv on the cell growth, hl 60 cells were incubated with different doses of rsv for 24 or 48 h, then cell viability was measured by mtt assay. Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. Cells are maintained in 10cm dishes at 37c and 4% co2 until 6080% confuency when confuent, cells are split 1. Treatment of this cell line with 1,25oh 2 d 3 results in a downregulation of expression of cmyc associated with cell differentiation 86. Cell culture was first successfully undertaken by ross harrison in 1907 roux in 1885 for the first time maintained. Maintenance 1 change media the day after seeding and every 23 days thereafter. Even this relatively insensitive method revealed large differences in. All further steps are described in the subculture section. From culture plates and cryovials to the proprietary claycomb media, look no further for a complete offering of products and services to support your research efforts. We recommend using cell number and volume for the assay and the negative control samples. Clone 15 hl60 atcc crl1964 homo sapiens peripheral blo.

The cell line was derived from a 36yearold woman who was originally reported. Hl60 cell avalanche transfection reagent human leukemia. Control of macrophage cell differentiation in human. The cells exhibit phagocytic activity and responsiveness to chemotactic stimuli. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. The hl60 cell line was derived from peripheral blood leukocytes of a. In a microbiological safety cabinet, hold a tissue soaked in 70% alcohol around the cap of the frozen ampoule and turn the cap a quarter turn to release any residual liquid nitrogen that may be trapped. The human promyelocytic leukemia cell line hl60 presents mul tiple genomic. Most cell lines can be grown using dmem culture media or rpmi culture media with 10% foetal bovine serum fbs, 2 mm glutamine and antibiotics can be added if required see table below. Perform a cell count to determine the viability of cells 3.