Colcemid Continues to Act Until the Step in the Harvest

October 13, 2022 Post a Comment

Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 4

Ngan F. Huang , ... Sunny Luke , in Handbook of Immunohistochemistry and in Situ Hybridization of Human Carcinomas, 2006

Materials

1.: Colcemid solution (10 μg/ml) (GIBCO)
2.: Cancer hypotonic solution: 3.0 g potassium chloride, 4.8 g HEPES (N-2-hybroxyethyl-piperazine-N-2-ethanesulfonic acid) (Sigma, St. Louis, MO), 0.2 g EGTA (ethylene-bis[oxyethylene nitrol] tetraacetic acid) (Sigma). (Dissolve in 1000 ml of distilled water and adjust pH to 7.0 using 10 N NaOH. Store at 4°C for 2–3 weeks.)
3.: 100 ml Trypsin-ethylenediamine tetraacetic acid (EDTA) solution (1X) (GIBCO)
4.: Fixative: 3 parts of methanol (Fisher), 1 part acetic acid (Fisher)
5.: Microscope slides
6.: Centrifuge tubes
7.: Pasteur pipettes
8.: Water bath at 37°C
9.: Incubator at 37°C
10.: Centrifuge
11.: Old media containing 10% FBS, or prepare new 1 ml of FBS with 9 ml of HBSS (GIBCO)
12.: Slide warmer

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DNA Repair Enzymes: Cell, Molecular, and Chemical Biology

Carolyn G. Marsden , ... Joann B. Sweasy , in Methods in Enzymology, 2017

3.4.2 Buffers and Reagents

•: MCF10A complete media
•: KaryoMax® Colcemid™ (10 μg/mL stock) (ThermoFisher)
•: Carnoy's fixative (3:1 methanol:acetic acid), prechilled to − 20°C
•: 1 × PBS
•: 0.05% trypsin
•: Potassium chloride (Gibco, 0.075 M), prewarmed to 37°C
•: Mounting media with DAPI (we use Sigma Fluoroshield™ with DAPI)
•: Cytochalasin B (Cyt-B) in ddH₂O
•: Methanol
•: Acridine orange in PBS
•: Ouabain octahydrate (Sigma)
•: 0.5% Crystal Violet (500 mg diluted in 100 mL 80% methanol)

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Adult Stem Cells

Candace L. Kerr , ... John D. Gearhart , in Methods in Enzymology, 2006

Cytogenetic Analysis

EGC colonies are incubated in growth medium with KaryoMAX Colcemid (0.1 μg/ml; Invitrogen) for 3–4 h at 37° in 5% CO₂ with 95% humidity, isolated from the feeder with a 1‐μl Pipetman and tip, and then disaggregated in 0.05% trypsin–EDTA at 37°. Cells are then resuspended in 2 ml of 0.75 M KCl hypotonic solution and incubated at 37° for 35 min. Next, cells are fixed by slowly adding 10 ml of cold Carnoy's fixative (methanol–acetic acid, 3:1) and rinsed twice with fixative before being plated. To prepare slides, ∼20‐μl cell suspensions are dropped onto microscope slides over a humidity chamber and then allowed to air dry. Metaphase chromosomes are stained in Giemsa staining solution (Invitrogen) and observed at ×600 magnification with oil immersion. Standard karyotyping includes approximately 20 metaphase spreads from each line to be examined for the presence of structural abnormalities of chromosomes and accurate chromosomal number.

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Volume 3

Carl W. Anderson , Ettore Appella , in Handbook of Cell Signaling (Second Edition), 2010

Microtubule Disruption

Activation of p53 also occurs in response to factors such as colcemid, nocodazole, and taxol that deregulate cell adhesion or microtubule architecture and dynamics. Taxol (Paclitaxel), one of the newer chemotherapy drugs commonly used to treat ovarian, breast, and head and neck cancers, inhibits microtubule depolymerization. After nocodazole treatment, which depolymerizes microtubules, quiescent human fibroblasts accumulated transcriptionally active p53 and arrested in G ₁ with a 4 N DNA content [191]. Activation of p53 after colcemid treatment was accompanied by a moderate increase in phosphorylation at Ser15 and correlated with activation of Erk1/2 MAP kinases and the development of focal adhesions rather than disruption of the microtubule system [192]. Curiously, murine fibroblasts did not undergo the same response. Taxol and vincristine, but not nocodazole, were found to induce multi-site phosphorylation of p53 in several tumor derived human cell lines, including HCT-116 and RKO cells, and the pattern of p53 phosphorylation was distinct from that observed after DNA damage [51, 193]. Nevertheless, both nocodozole and taxol increased phosphorylation at Ser15 (Figure 264.2). Interestingly, microtubule inhibitor induced p53 stabilization and Ser15 phosphorylation did not occur in ATM deficient fibroblasts, nor in normal human dermal fibroblasts. Studies with ectopically expressed p53 phosphorylation site mutants indicated that several p53 amino-terminal residues, including Ser15 and Thr18, were required for the taxol mediated phosphorylation of p53 [193]. In contrast, Damia et al. [194] reported taxol induced p53 phosphorylation at Ser20 but not at Ser15 in HCT-116 cells. Ser20 phosphorylation was accompanied by increased Chk2 activity and was inhibited neither in A-T cells lines nor by wortmannin treatment. Thus, the signaling pathways that impinge on p53 after hypoxia, ribonucleotide depletion, or microtubule disruption, while still not well defined, appear distinct from the pathways induced by genotoxic stresses.

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Classical Cytogenetics

Marlys Houck , in Human Stem Cell Manual (Second Edition), 2012

Reagents and Supplies

Item	Supplier	Catalog no.	Alternative
Acetic Acid, Glacial	Gallade Chemical, Inc.	2504-14	Many
Colcemid, KaryoMax® (Gibco)	Life Technologies	15212-012
Coplin jars	VWR	25460-000	Many
Coverslips no. 1	VWR	48393-081	Many
Gurr's Buffer Tablets	Life Technologies	10582-013
Gurr's Giemsa Stain	CTL Scientific Supply Corp	35086HE	Many
Harleco Wright-Giemsa stain, EM Science	VWR	15204-232
Methyl Alcohol, Anhydrous (99.8% min.) (Mallinckrodt Chemicals) (methanol)	Gallade Chemical, Inc.	301606	Many
Microcentrifuge tubes, 1.5 mL	VWR	20170-355	Many
Mounting Medium	VWR	48212-290	Many
Mounting Medium with DAPI	Vector Laboratory	H-1200
Pasture pipettes 5 glass	VWR	14672-608	Many
Pipettes: 5 mL, 10 mL	Corning	4487,4488	Many
Potassium Chloride (KCl)	Fisher Scientific	P217-500	Many
Slides, microscope Fisherbrand Superfrost CyGen	Fisher Scientific	22-034-730
Trypsin-EDTA (TE) for cell culture, Gibco	Life Technologies	25300-062	TrypLE Express™ Cat.#12604
Trypsin 250 (for banding)	Becton Dickinson	215240

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Advances in Radiation Biology

Andrew R.S. Collins , Robert T. Johnson , in Advances in Radiation Biology, 1984

E Repair Activity in Relation to the Cell Cycle

A further application of inhibitors of repair has been in the analysis of incision capacity at different points in the cell cycle. CHO cells snychronized by mitotic selection with colcemid were followed for 9 hr into interphase, i.e., to mid S phase ( Collins et al., 1980). DNA break accumulation during 30-min incubation after irradiation with a wide range of UV doses was assayed, under various inhibitory conditions. A combination of HU and ara C, with preincubation, gave the greatest accumulation of breaks. Ara C alone gave a lower break frequency at a given UV dose. The potentiating effect of HU was most marked in mitosis and S phase, in which—at least at doses below 10 J m⁻²—ara C alone caused virtually no accumulation of breaks. The varying effectiveness of inhibitors during the cycle is probably related to the varying concentrations of DNA precursors in the cellular pool. S phase CHO cells contain the highest concentration of dCTP (Skoog et al., 1973; Walters et al., 1973), and in these cells (1) the effect of ara C alone is relatively small, and (2) the potentiation of this effect by HU is large (Collins et al., 1980). Cleaver (1981), too, found that ara C alone had a minimal effect on repair in S phase, which he accounts for as the result of a large competing dCTP pool. However, whereas Cleaver found that even with HU (2 × 10⁻³ M) present too, breaks accumulated to a much lower level in S phase DNA than in non-S phase DNA, we found that HU (10⁻³ M) and ara C together cause similar levels of breakage in G₁ and S phase. By contrast, the level of accumulation of breaks in mitotic cells was low, even with both inhibitors present.

We summarized these data, representing incision during the 30-min incubation period, by calculating values of K _m and V _max for the different points in the cycle and, also, the number of breaks made per dimer in 30 min at infinitely low dimer concentration. (Because these hamster cells incise at a much lower rate than normal human cells, it is not necessary to calculate precisely the initial rate of incision in order to estimate these parameters.) Figure 19 shows that between early G₁ and mid-S phase there is no significant variation in any of the parameters of incision, but there are great differences in all three between mitosis and interphase. Mitotic cells show a relatively high K _m (i.e., a low affinity of enzyme for damaged DNA, most likely resulting from the highly condensed nature of mitotic chromatin rendering the dimers inaccessible) and a low V _max (indicating less enzyme available per unit DNA, or a longer time taken for endonuclease molecules to act on DNA damage and be released for action elsewhere, perhaps, again, as a result of the extreme condensation of chromatin in mitosis). The third parameter, representing the activity at infinitely low dimer concentration, is of interest because very low UV doses are the biological norm. In the case of CHO cells, the parameter varies in a similar way to V _max: mitotic cells given very low doses of irradiation can incise at only 1 in 50 dimers in 30 min, whereas interphase cells can process 1 in 4.

The pattern of repair activity seems to be different in cells passing through the synchronous cell cycle following release from the quiescent, nondividing state. We examined incision in Microtus agrestis cells (Fig. 20; Downes et al., 1982), and found an immediate rise in V _max following trypsinization of quiescent cultures and replating at lower density. V _max falls sharply as cells enter S phase. This pattern is similar to the peak of UV-induced UDS seen in late G₁ in normal human cells released from quiescence (Gupta and Sirover, 1981). K _m also rises as the cells approach S phase, and then falls. Thus, as G₁ progresses, the amount of enzyme available (reflected in V _max) increases but its affinity for damaged DNA (reflected in K _m) declines. These antagonistic changes are in contrast to the coordinated change in CHO cells from low V _max, high K _m in mitosis to high V _max, low K _m in interphase. At low (biologically relevant) UV doses in Microtus, the K _m change predominates, and this is illustrated by the low-dose effectiveness of incision (i.e., breakage rate per dimer at infinitely low dimer concentration), which falls steadily through G₁ to a minimum in S phase (Fig. 20).

After the first cell cycle, V _max and K _m stabilize at relatively low levels, similar to those for randomly proliferating Microtus cells (also shown in Fig. 20). At 60 hr after release, when the labeling index indicates approach to a quiescent state, V _max has risen to its starting value of about four breaks per 10⁹ daltons in 30 min.

These alterations in incision capacity in cells at different growth states should be taken into account when assessing the responses of mammalian cells to DNA damage. Cells held at quiescence are capable of repairing potentially lethal UV damage (Konze-Thomas et al., 1979; Simons, 1979; Downes et al., 1982); cells exposed to UV at different times in the cell cycle show different levels of survival (reviewed in Downes et al., 1979) and mutagenesis (Burki et al., 1980); and comparisons of repair and other responses to damage in different cell types may be invalid unless growth states are identical, or the variation in repair with cell state is known and allowed for.

A novel use of HU as a repair inhibitor in cell cycle studies has recently been reported (Ben-Hur et al., 1982). Stimulation of plateau-phase human cells into growth by a medium change results in induction of ornithine decarboxylase activity to a peak about 5 hr later. The induction is transcriptionally controlled, and damage to DNA (e.g., by UV irradiation) inhibits it. Incubation of cells for 20 hr after UV irradiation before giving the induction stimulus restores the induction of the enzyme. Ben-Hur et al. (1982) confirmed that the recovery was the result of excision repair of the DNA damage by incubating the cells with HU during the recovery period after UV. In this case, induction of the enzyme was again absent, presumably because, HU having inhibited repair, the DNA damage remained as a block to transcription.

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Harlequin Chromosomes☆

G. Ludewig , S. Flor , in Reference Module in Life Sciences, 2017

Protocol to Obtain Harlequin Chromosomes

A detailed protocol for the preparation of harlequin chromosomes could be as follows: cells are grown in the presence of BrdU for a period of time sufficient to pass through two cell cycles; Colcemid or another spindle poison may be added during the final hours to arrest cells in metaphase. Cells are harvested, in case of adhered cells by using trypsin or a similar enzyme for detaching them, washed with phosphate-buffered saline (PBS), and briefly incubated with a hypotonic solution to swell the cells which will facilitate the spreading of chromosomes on the microscope slide. The cells are then fixed by dropwise, to avoid clumping of cells, adding a solution of ice-cold glacial acetic acid:methanol (1:3, v/v) to the PBS-cell suspension. The fixative is changed twice, and the cells are stored at −20°C for at least 24 h. After replacing the fixative with enough freshly prepared fixative to achieve a denser cell suspension, cells are dropped on microscope slides to prepare metaphase spreads. Samples are then air-dried and "aged" for at least 2 days. For the FPG staining, slides are immersed into Hoechst 33258 solution, then mounted with PBS and exposed to UV light, followed by exposure to SSC buffer at 60°C, and finally stained with Giemsa. The resulting metaphase spreads of harlequin chromosomes can be analyzed by light microscopy. Typically, the number of chromosomes per metaphase and the number of switches of the dark–light staining from one chromatid to the other, that is, the number of SCEs, in all chromosomes of the metaphase are counted.

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The In Vitro Chromosome Aberration Test

Marilyn Registre , Ray Proudlock , in Genetic Toxicology Testing, 2016

7.5 Consumables and Reagents

Consumables typically used in a testing laboratory (sterile where appropriate) include:

1.: Blood collection tubes with sodium heparin ²
2.: Centrifuge tubes, 15 and 50 mL polypropylene with caps
3.: Colchicine or colcemid
4.: Coverslips, 22×50 mm
5.: Culture medium. RPMI 1640 is used for lymphocytes and is available commercially in various forms: powder, 10× and 1× liquid, and as an autoclavable solution. The 1× liquid can usually be used as is, but other forms will need supplementing with sodium bicarbonate and/or glutamate if these are not already present. Some forms of RPMI (Dutch modification) include HEPES as a buffering agent, but this tends to be inhibitory and is best avoided. (Ham's) F-12 medium is used for CHO cells
6.: Culture vessels: 25 and 75 cm² vented flasks (used for adhesive cell lines) ³ ; vials, tubes, or plates as appropriate, such as 4 mL clear, glass, flat-bottom, screw cap vials or flat-sided culture tubes (Nunc or equivalent) for 1 and 5 mL blood cultures, respectively ²
7.: Dulbecco's phosphate-buffered saline (DPBS) without calcium or magnesium ³
8.: Fetal calf serum (FCS)/fetal bovine serum (FBS)
9.: Filter papers, Whatman No. 1 to fit Büchner funnel (or cotton wool)
10.: Filters units, 0.2 or 0.22 µm Luer-Lok syringe-fitting for aqueous solutions and solvents, such as Millex®
11.: Fix—3 volumes methanol:1 volume glacial acetic (ethanoic) acid prepared just before each use
12.: Gentamicin, aqueous 10 mg/mL (commercially available)
13.: Hypotonic—0.075 M potassium chloride
14.: Immersion oil for microscopy
15.: Isoton II diluent for cell counting with the Coulter counter ³
16.: Labels, self-adhesive printed with code number—code numbers can be generated in Excel using the random function and then printed onto sheets of adhesive labels with an appropriate number of replicate labels per culture. The slide code should be printed and saved electronically to decode results later.
17.: Medical wipes, such as Kimwipes
18.: Microcentrifuge tubes
19.: Micropipette tips, sterile
20.: Pasteur pipettes—the polypropylene ones are most convenient
21.: Phosphate buffer, 0.2 M, pH 7.4
22.: Pipettes, sterile plastic disposable
23.: Positive control agents
24.: Purified water
25.: Results sheets/forms
26.: S9 fraction and cofactors
27.: Slide mountant (Cytoseal, DPX, Permount, or similar permanent nonaqueous type)
28.: Slide trays, cardboard
29.: Solvents including appropriate anhydrous organic solvents; DMSO in particular is hygroscopic and can develop mutagenic impurities in the presence of small amounts of water. Pure organic solvents should be maintained in an anhydrous condition by addition of a small quantity of a compatible predried molecular sieve (type 4A in the case of DMSO) and stored well-sealed over anhydrous silica gel.
30.: Stain, Giemsa solution in methanol/glycerol (see recipe in the rodent micronucleus chapter). Giemsa stain Gurr solution can also be purchased from VWR and Fisher.
31.: Syringes, disposable
32.: Trypan blue, 0.4% solution in DPBS
33.: Trypsin 0.25% in DPBS (with or without 1 mM EDTA) ³

Gas syringes or metering equipment, 24 mL glass anaerobic culture tubes (Bellco Glass), gas-impermeable injectable butyl rubber septum, gas bags, sealable vials, and other specialist equipment or components will be needed to test gases.

See the next section (Reagents and Recipes) for additional components and reagents that may be needed.

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Breakage Analysis and DEB Testing

Peter Mudiaga Etaware , in Encyclopedia of Infection and Immunity, 2022

Fibroblast tissues

In the absence of peripheral blood, DEB test can be performed on fibroblast cultures i.e., initiated from skin biopsies, excised pieces of lungs or skin fragments from an abortus or a stillborn etc.

Laboratory requirements:

•: Fibroblast tissues.
•: Complete DMEM medium (Supplemented with 20% FBS).
•: PBS.
•: Diepoxybutane (1,3-butadiene diepoxide), store at 4 °C.
•: 1 μg/mL Colcemid (store at 4 °C).
•: Fixative i.e., 3:1 (v/v) methanol/glacial acetic acid (freshly prepared).
•: 0.051 M (0.38% w/v) KCl.
•: Tissue culture plates (60 mm capacity).
•: Disposable scalpels.
•: Punch (3 mm).
•: Additional reagents and equipment for cell passaging and counting.

Procedure:

Collect fibroblast tissue with the aid of a 3 mm punch. Place the fibroblast tissue in 0.5 mL complete DMEM medium (fortified with 20% FBS) and cut to standard fragment (1 × 1 mm) using two sterile disposable scalpels, in a 60 mm tissue culture plate. Draw horizontal and vertical lines across the inner surface of three (3) sterile disposable tissue culture plates (60 mm capacity) using the pointed edge of a sterile scalpel. Transfer 5–6 excised pieces of fibroblast tissues into each of the 60 mm culture plate. Place the tissues at the intersect of the scored lines. The score lines will facilitate easy attachment of tissue fragments to the culture plates and also, tracing of tissue colonies. Incubate the scored plates, without any additional tissue culture medium, at 37 °C for 15–30 min, to allow the pieces of tissue to adhere to the plates. Afterwards, add 5 mL of complete DMEM medium (with 20% FBS) to each plate and incubate the cultures at 37 °C, with 5% CO₂. Replace the culture medium every 3–4 days without disturbing the tissue fragments. Use an inverted microscope to inspect the cultures for evidence of cell growth.

As soon as a huge patch of new fibroblasts cells surrounds the tissue fragments in the plate, carry out cell passaging and trypsinization by treating both the proliferated fibroblast cells and initial tissue fragments with 1 × trypsin/EDTA solution i.e., remove the medium in which the newly proliferated fibroblast cells were suspended by decanting the liquid without agitating the cell clusters, wash the monolayered-cell clusters with a reasonable quantity of saline solution, free of Ca²⁺ and Mg²⁺. Thereafter, remove the saline solution from the culture by decanting or with the aid of a Pasteur pipette. Add excess trypsin/EDTA solution to the culture such that the monolayers of cell clusters are completely submerged by the solution. Incubate the proliferated cell culture at 37 °C for 2 min in a mist of 5% CO₂. Afterwards, aseptically remove the trypsin/EDTA solution by decanting (without agitation) and incubate the residual cells at 37 °C until cells start to detach from the surface of the monolayer cluster. The progress of cell detachment can be monitored intermittently by the use of an inverted microscope. Extreme care should be taken not to dislodge the cell colonies formed, as they will continue to proliferate in the culture, if undisturbed.

If there are more than enough fibroblast cells formed within any of the tissue culture flask, transfer some of the newly generated cells to a sterile tissue culture flask (25 mL capacity) submerged in complete DMEM/15% FBS solution (Note: reduce the concentration of FBS in the DMEM medium to 15% for this and all subsequent subcultures). These cells are designated as "first passage cells." Add 1 × trypsin/EDTA solution when the first passage culture reaches convergence. Subculture at a 1:3 split ratio in complete DMEM/15% FBS. Cell passaging and trypsinization can be conducted until there are enough proliferated cells for the experiment. You can finally proceed with DEB treatment when at least three "second passage cultures" reach convergence. Plate the proliferated fibroblast cells in 5 mL complete DMEM medium (fortified with 15% FBS) in 25 mL capacity tissue culture flask (3 × 10⁵ cells per flask). Incubate cultures at 37 °C, with 5% CO₂, for 24 h.

Prepare the required DEB treatment by adding 10 mL of PBS to test tube A, 6 mL to test tube B, and 4.5 mL to test tubes C and D, respectively. Furthermore, add 10 μL of DEB solution (fortified with 15% FBS) to test tube A, cap, and mix gently. Transfer 4 mL from test tube A to B, cap, and homogenize the content by shaking manually, then aseptically transfer 0.5 mL (500 μL) from test tube B to C, cap, and mix gently too. Complete the dilution process by pipetting 0.5 mL (500 μL) from test tube C to D, homogenize the content manually and allow to stand for a few seconds. Prepare a blank treatment in test tube E (using sterile distilled water).

After incubation, pipette 25 μL of the treatment solution from test tube C into three (3) out of the nine (9) incubated Fibroblast cells, label the tissue culture plates as C1, C2 and C3, respectively. Repeat the procedure using test tube D and three (3) out of the six (6) remaining cultures and label the tissue culture plates as D1, D2 and D3. Finally, pipette 25 μL of the "blank treatment" from test tube E into the remaining cultures and label the tissue culture plates as E1, E2 and E3, respectively. The final DEB concentrations in the treated tissue culture plates would be 0.10 μg/mL (C1, C2 and C3), 0.01 μg/mL (D1, D2 and D3) and 0.00 μg/mL (E1, E2 and E3), respectively. Afterwards, incubate all the tissue culture plates at 37 °C, under a controlled cloud of 5% CO₂, for 48–72 h. Add 2 mL of 1 μg/mL Colcemid to each of the treated tissue culture plates and incubate further for another 20 min. Transfer the content of each of the tissue culture plates (both treated and untreated) to separate sterile conical-bottom centrifuge tubes (15 mL capacity each), cap, and centrifuge at 800–1000 rpm (in an IEC clinical centrifuge) for 10 min. Decant the supernatant, retaining about 0.5 mL of liquid, re-suspend the cell pellets at the bottom of the tubes in the leftover supernatant (0.5 mL) by flipping each tube manually (to aide homogenization). Add 5 mL of pre-warmed 0.075 M KCl and incubate for another 10 min at 37 °C, in a water bath.

Centrifuge again at 1000 rpm for 10 min. Completely remove the supernatant and gently add 1 mL of freshly prepared fixative without disturbing the cell pellets. Decant to remove fixative immediately (after 3–5 s) and repeat this procedure two more times. Afterwards, tap the tube gently to break up the pellets. Instantaneously, add fixative to re-suspend the cells without agglutination or cluster formation. Step up the volume to 8–10 mL with fixative. Break up any clumps by pipetting with a Pasteur pipet. Allow to stand for 30 min at 25 °C. At the end of 30 min, centrifuge at 1000 rpm for 10 min. completely decant the supernatant and re-suspend the cell pellets in about 3–5 mL of fresh fixative. Allow to stand for another 10 min. Repeat this step once or twice, immediately or after a window period of 24 h.

Harvest cultures at the first mitotic stage (precisely, at the metaphase stage) after subculture (between 24 and 48 h). Add 1 mL of 1 μg/mL Colcemid (per 5 mL medium) to all the tissue culture plates, at 3 h prior to harvest. Incubate for another 3 h at 37 °C, with 5% CO₂. The timing for harvesting is determined by the intermittent inspection of the cultures with an inverted microscope. Metaphase cells appear rounded. Treat the fibroblast cells again with 1 × trypsin/EDTA solution (use the predefined trypsinization procedure). Transfer the contents of each tissue culture plates to a sterile 15 mL centrifuge tube, cap, and centrifuge for 10 min at 1000 rpm. Decant, retaining about 0.5 mL of liquid, re-suspend the cell pellets at the bottom of the tubes in the leftover supernatant (0.5 mL) by flipping each tube manually (to aide homogenization), with the careful addition of 5 mL of pre-warmed 0.075 M KCl. Incubate the mixture for 10 min at 37 °C, in a water bath. Afterwards, centrifuge at 1000 rpm for 10 min. Fix the fibroblast cells as described above for blood samples (Auerbach, 2015). Note: The hypotonic solution of KCl applied to the fibroblast cells should be more diluted than that used for peripheral blood cultures.

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The Epigenetics of Endometriosis

Sun-Wei Guo , in Epigenetics in Human Disease, 2012

22.5.2 "Reader/Effector" Modules

Histone modifications are recognized by "reader/effector" modules, which read and interpret modification codes or marks and then execute conformational changes in chromatins and provide signals to regulate chromatin dynamics. These modules include Plant Homeo Domain (PHD), chromo (for lysine methylation), bromo (for lysine acetylation), tudor, proline-tryptophan-tryptophan-proline (PWWP), SWI3p-Rsc8p-Moira (SWIRM), SWI3-ADA2-N-CoR-TFIIIB (SANT), and Malignant Brain Tumor (MBT) domains. By recruiting these reader/effector proteins, histone modifications lead to changes in chromatin structure as well as dynamics [111]. As at time of writing, there has been no published account on aberration of any "reader/effector" modules in histone modifications.

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Colcemid Continues to Act Until the Step in the Harvest

Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 4

Materials

DNA Repair Enzymes: Cell, Molecular, and Chemical Biology

3.4.2 Buffers and Reagents

Adult Stem Cells

Cytogenetic Analysis

Volume 3

Microtubule Disruption

Classical Cytogenetics

Reagents and Supplies

Advances in Radiation Biology

E Repair Activity in Relation to the Cell Cycle

Harlequin Chromosomes☆

Protocol to Obtain Harlequin Chromosomes

The In Vitro Chromosome Aberration Test

7.5 Consumables and Reagents

Breakage Analysis and DEB Testing

Fibroblast tissues

The Epigenetics of Endometriosis

22.5.2 "Reader/Effector" Modules

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