We thank Jessica Hall for her valuable discussion.

1. Preparation of Primary Insect Spermatocyte Cell Culture for Micromanipulation
<ol>
	<li>Slide preparation
	<ol>
		<li>Obtain a 75 mm x 25 mm glass slide with a 20 mm diameter circular hole cut out in the center of the slide. 
		NOTE: These were cut from a single sheet of window glass to be the size of a glass slide with a hole in the center.</li>
		<li>Run a 25 mm x 25 mm #1.5 coverslip through a Bunsen-burner flame for 2 s.</li>
		<li>Apply vacuum grease around the edge of the hole in the glass slide</li>
		<l...

<ol>
	<li>Chambers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+studies+II.+The+cell+aster:+a+reversible+gelation+phenomenon.">Microdissection studies II. The cell aster: a reversible gelation phenomenon.</a> Journal of Experimental Zoology. 23 (3), 483-505 (1917).</li><li>Carlson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+studies+of+the+dividing+neuroblast+of+the+grasshopper,+Chortophaga+viridifasciata.">Microdissection studies of the dividing neuroblast of the grasshopper, Chortophaga viridifasciata.</a> Chromosoma. 5 (3), 199-220 (1952).</li><li>Nicklas, R. B., Staehly, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation.+I.+The+mechanics+of+chromosome+attachment+to+the+spindle.">Chromosome micromanipulation. I. The mechanics of chromosome attachment to the spindle.</a> Chromosoma. 21 (1), 1-16 (1967).</li><li>Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation.+II.+Induced+reorientation+and+the+experimental+control+of+segregation+in+meiosis.">Chromosome micromanipulation. II. Induced reorientation and the experimental control of segregation in meiosis.</a> Chromosoma. 21 (1), 17-50 (1967).</li><li>Nicklas, R. B., Koch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation.+3.+Spindle+fiber+tension+and+the+reorientation+of+mal-oriented+chromosomes.">Chromosome micromanipulation. 3. Spindle fiber tension and the reorientation of mal-oriented chromosomes.</a> Journal of Cell Biology. 43 (1), 40-50 (1969).</li><li>Nicklas, R. B., Ward, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+of+error+correction+in+mitosis:+microtubule+capture,+release,+and+tension.">Elements of error correction in mitosis: microtubule capture, release, and tension.</a> Journal of Cell Biology. 126 (5), 1241-1253 (1994).</li><li>Li, X., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+forces+control+a+cell-cycle+checkpoint.">Mitotic forces control a cell-cycle checkpoint.</a> Nature. 373 (6515), 630-632 (1995).</li><li>Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+the+force+produced+by+the+mitotic+spindle+in+anaphase.">Measurements of the force produced by the mitotic spindle in anaphase.</a> Journal of Cell Biology. 97 (2), 542-548 (1983).</li><li>Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+forces+that+move+chromosomes+in+mitosis.">The forces that move chromosomes in mitosis.</a> Annual Review of Biophysics and Biophysical Chemistry. 17, 431-449 (1988).</li><li>Nicklas, R. B., Koch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation.+IV.+Polarized+motions+within+the+spindle+and+models+for+mitosis.">Chromosome micromanipulation. IV. Polarized motions within the spindle and models for mitosis.</a> Chromosoma. 39 (1), 1026 (1972).</li><li>Zhang, D., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+chromosomes+and+centrosomes+on+spindle+assembly+as+observed+in+living+cells.">The impact of chromosomes and centrosomes on spindle assembly as observed in living cells.</a> Journal of Cell Biology. 129 (5), 1287-1300 (1995).</li><li>Nicklas, R. B., Lee, G. M., Rieder, C. L., Rupp, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+cut+mitotic+spindles:+clean+cuts+and+stable+microtubules.">Mechanically cut mitotic spindles: clean cuts and stable microtubules.</a> Journal of Cell Science. 94 (Pt 3), 415-423 (1989).</li><li>Zhang, D., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomes+initiate+spindle+assembly+upon+experimental+dissolution+of+the+nuclear+envelope+in+grasshopper+spermatocytes.">Chromosomes initiate spindle assembly upon experimental dissolution of the nuclear envelope in grasshopper spermatocytes.</a> Journal of Cell Biology. 131 (5), 1125-1131 (1995).</li><li>Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+distribution:+experiments+on+cell+hybrids+and+in+vitro.">Chromosome distribution: experiments on cell hybrids and in vitro.</a> Philosophical Transactions of the Royal Society of London B. 227 (955), 267-276 (1977).</li><li>Paliulis, L. V., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reduction+of+chromosome+number+in+meiosis+is+determined+by+properties+built+into+the+chromosomes.">The reduction of chromosome number in meiosis is determined by properties built into the chromosomes.</a> Journal of Cell Biology. 150 (6), 1223-1232 (2000).</li><li>Church, K., Nicklas, R. B., Lin, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulated+bivalents+can+trigger+mini-spindle+formation+in+Drosophilamelanogaster+spermatocyte+cytoplasm.">Micromanipulated bivalents can trigger mini-spindle formation in Drosophilamelanogaster spermatocyte cytoplasm.</a> Journal of Cell Biology. 103 (6), 2765-2773 (1986).</li><li>Forer, A., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+autosome+movements+and+of+sex-chromosome+movements+on+sex-chromosome+segregation+in+crane+fly+spermatocytes.">Influence of autosome movements and of sex-chromosome movements on sex-chromosome segregation in crane fly spermatocytes.</a> Chromosoma. 40 (4), 417-442 (1973).</li><li>Camenzind, R., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+non-random+chromosome+segregation+in+spermatocytes+of+Gryllotalpa+hexadactyla.+A+micromanipulation+analysis.">The non-random chromosome segregation in spermatocytes of Gryllotalpa hexadactyla. A micromanipulation analysis.</a> Chromosoma. 24 (3), 324-335 (1968).</li><li>Ault, J. G., Felt, K. D., Doan, R. N., Nedo, A. O., Ellison, C. A., Paliulis, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-segregation+of+sex+chromosomes+in+the+male+black+widow+spider+Latrodectus+mactans+(Araneae,+Theridiidae).">Co-segregation of sex chromosomes in the male black widow spider Latrodectus mactans (Araneae, Theridiidae).</a> Chromosoma. 126 (5), 645-654 (2017).</li><li>Felt, K. D., Lagerman, M. B., Ravida, N. A., Qian, L., Powers, S. R., Paliulis, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+the+amphitelically+attached+univalent+X+chromosome+in+the+spittlebug+Philaenus+spumarius.">Segregation of the amphitelically attached univalent X chromosome in the spittlebug Philaenus spumarius.</a> Protoplasma. 254 (6), 2263-2271 (2017).</li><li>Golding, A. E., Paliulis, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype,+sex+determination,+and+meiotic+chromosome+behavior+in+two+pholcid+(Araneomorphae,+Pholcidae)+spiders:+implications+for+karyotype+evolution.">Karyotype, sex determination, and meiotic chromosome behavior in two pholcid (Araneomorphae, Pholcidae) spiders: implications for karyotype evolution.</a> PLoS One. 6, e24748 (2011).</li><li>Doan, R. N., Paliulis, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+reveals+an+XO-XX+sex+determining+system+in+the+orb-weaving+spider+Neoscona+arabesca+(Walckenaer).">Micromanipulation reveals an XO-XX sex determining system in the orb-weaving spider Neoscona arabesca (Walckenaer).</a> Hereditas. 146 (4), 180-182 (2009).</li><li>Paliulis, L. V., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+chromosomes+reveals+that+cohesion+release+during+cell+division+is+gradual+and+does+not+require+tension.">Micromanipulation of chromosomes reveals that cohesion release during cell division is gradual and does not require tension.</a> Current Biology. 14 (23), 2124-2129 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology Micromanipulator. DIY High Precision Micromanipulator. , (2018).</li><li>Ellis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezoelectric+micromanipulators.">Piezoelectric micromanipulators.</a> Science. 138 (3537), 84-91 (1962).</li><li>Ellis, G. W., Begg, D. A., Zimmerman , . A. M., Forer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation+studies.">Chromosome micromanipulation studies.</a> Mitosis/Cytokinesis. , 155-179 (1981).</li><li>Powell, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microforge+attachment+for+the+biological+microscope.">A microforge attachment for the biological microscope.</a> Journal. Royal Microscopical Society. 72 (4), 214-217 (1953).</li><li>Alsop, G. B., Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+continuously+dictate+distribution+of+actin+filaments+and+positioning+of+cell+cleavage+in+grasshopper+spermatocytes.">Microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes.</a> Journal of Cell Science. 117 (Pt 8), 1591-1602 (2004).</li><li>Zhang, D., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphase'+and+cytokinesis+in+the+absence+of+chromosomes.">Anaphase' and cytokinesis in the absence of chromosomes.</a> Nature. 382, 466-468 (1996).</li></ol>

With practice, moving chromosomes around in the cell can become second nature. Needles that are both sufficiently stiff and sufficiently thin-tipped are difficult to &#34;get the knack of&#34; fabricating, but this ability also comes with practice. Needles that are so fine that they deform when moved in the halocarbon oil will not be useful for pushing chromosomes in the cell. Needles that are so blunt that their tips are visible and as large as 1/3 of the width of a chromosome (or larger) are very likely to kill the cel...

Micromanipulation has revealed parts of the mechanism for a chromosome congression, the spindle checkpoint, and anaphase chromosome movements. The earliest publication describing the results of micromanipulation experiments was by Robert Chambers1. Chambers used a mechanical micromanipulator with an attached glass needle to probe the cytoplasm of a number of different cell types. Unfortunately, contrast methods that allowed the visualization of chromosomes and many other cellular components in living cells were not available at the time, so Chambers&#39; experiments could not show the effects of repositioning such cellular components. Early micromanipulations that altered the chromosome position used the Chambers apparatus to sweep the spindle midzone in anaphase cells, showing that such manipulations could alter the position of chromosome arms in anaphase grasshopper neuroblasts2. Nicklas and his collaborators were the first to perform fine micromanipulations of chromosomes, stretching the chromosomes3, detaching them from the spindle and inducing a reorientation3,4, stabilizing a malorientation by applying tension to the chromosomes5,6,7, and measuring the forces produced by spindles in anaphase8,9. Other work by the Nicklas lab showed that cytoplasmic granules could also be manipulated10 and that centrosomes could be repositioned by micromanipulation11. Micromanipulation is not just useful for moving chromosomes and other cellular components. A micromanipulation needle can cleanly cut through a mitotic spindle in demembranated cells12 or can be used to dissolve the nuclear envelope13. In addition, adjacent cells can be fused by micromanipulation14,15.
With such a wide variety of interesting experiments that can be done using micromanipulation, it is at first glance surprising that micromanipulation experiments have been done by very few chromosome biologists. One reason for this deficiency is that the mitotically-dividing cultured cells that are derived from vertebrate tissues and are commonly used for studying chromosome movements are extremely difficult to micromanipulate. These tissue culture cells generally have a cortical cytoskeleton that &#34;gets in the way&#34; of the micromanipulation needle, and chromosomes either cannot be reached by the needle or the needle grinds through the cell, leading to a cell rupture and death. We, and other experimenters who use micromanipulation, have found arthropod cells to be amenable to micromanipulation. Arthropod spermatocytes are easily spread under a layer of halocarbon oil and appear to have a much less robust cortical cytoskeleton underlying the cell membrane during a cell division. Thus, arthropod testes provide a good source of meiotically-dividing cells (spermatocytes) and mitotically-dividing cells (spermatogonia) with easily accessible chromosomes for micromanipulation. A serial sectioning of a grasshopper spermatocyte fixed during a manipulation revealed that the needle never penetrates the cell membrane; the cell membrane deforms around the needle (Nicklas R.B., personal communication). Spermatocytes from a number of insect and spider taxa have been micromanipulated successfully, including grasshoppers, praying mantids, fruit flies, crane flies, crickets, spittlebugs, moths, black widow spiders, cellar spiders, and orb-weaving spiders3,7,17,18,19,20,21,22. Cultured, mitotically-dividing cells from insects can be micromanipulated. For example, the chromosomes in grasshopper neuroblasts in a primary culture have chromosomes that can be readily micromanipulated2,23. We suspect that the available cultured lines derived from Drosophila and other insects will also be micromanipulatable, though we have not tested the technique with these cells. We will show how dividing cells from grasshoppers and crickets can be prepared for a micromanipulation. Crickets are easy to obtain from most pet stores at any time of the year. Grasshoppers are only easily obtainable in the summer unless the researcher has access to a laboratory colony, but the species used (Melanoplus sanguinipes) has easily flattened cells, and long, easy-to-manipulate chromosomes.
Another reason why micromanipulation experiments have been done by a small handful of biologists is that micromanipulators that move chromosomes well are rarely available in the marketplace. We have found that a joystick-controlled piezoelectric micromanipulator controls the needle movement with no vibration, drift, or lag between the joystick movement and the needle movement, but other types of manipulators can also successfully push chromosomes around in the cell. The micromanipulators designed by Ellis and Begg25,26 are ideal for the micromanipulation of chromosomes, though they use older technology. Piezoelectric micromanipulators are currently available and commonly used in electrophysiology; however, these micromanipulators are not typically joystick-controlled. Joystick control is key to the smooth movements required for a successful micromanipulation, and so a custom joystick should be constructed to make the currently-available piezoelectric micromanipulators work for a chromosome micromanipulation. The joystick-controlled piezoelectric micromanipulators that work best have direct position control, in which the movement of the joystick translates directly to a needle movement.
A newly-designed piezoelectric micromanipulator can be constructed from commercially-available parts that can be easily replaced and from some small 3-D printed components, and it works well for chromosome micromanipulation24. The micromanipulator has adjustable sensitivity, manual coarse positioning, and no vibration, drift, or lag in the needle movement, and direct position control of the needle. Scientists can construct the micromanipulator using instructions available online24. Below are the methods for preparing a primary spermatocyte cell culture and for micromanipulating the chromosomes within the cells in that culture.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">VWR micro cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48366 249</td>
			<td style="width:167px;">25x25 mm, no 1.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dow Corning High Vacuum Grease</td>
			<td style="width:71px;">VWR</td>
			<td>AA44224-KT</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">KEL-F Oil #10</td>
			<td style="width:71px;">Ohio Valley Specialty Chemical</td>
			<td align="right" style="width:119px;">10189</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microdissecting Scissors, Stainless Steel</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>S3271-1EA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dumont #5 fine foreceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">0.85 mm outer diameter, 0.65 mm inner diameter Pyrex glass tube&#160;</td>
			<td style="width:71px;">Drummond Scientific</td>
			<td style="width:119px;">Custom order--call to request</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Inverted, Phase contrast microscope with 10X or 16X low magnification objective and 60X or 100X high magnification objective</td>
			<td style="width:71px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">microforge</td>
			<td style="width:71px;">either custom built or Narashige</td>
			<td style="width:119px;">MF-900</td>
			<td></td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">micromanipulator</td>
			<td style="width:71px;">either custom built or Burleigh PCS-6000 with custom piezo-controlling joystick</td>
			<td style="width:119px;">PCS-6300</td>
			<td></td>
		</tr>
	</tbody>
</table>

micromanipulation of chromosomes in insect spermatocytes

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase chromosome movements, and has been key to understanding what controls chromosome movements during a cell division. A skilled biologist can use a micromanipulator to detach chromosomes from the spindle, to reposition chromosomes within the cell, and to apply forces to chromosomes using a small glass needle with a very fine tip. While perturbations can be made to chromosomes using other methods such as optical trapping and other uses of a laser, to date, no other method allows the repositioning of cellular components on the scale of tens to hundreds of microns with little to no damage to the cell.
The selection and preparation of appropriate cells for the micromanipulation of chromosomes, specifically describing the preparation of grasshopper and cricket spermatocyte primary cultures for the use in live-cell imaging and micromanipulation, are described here. In addition, we show the construction of a needle to be used for moving chromosomes within the cell, and the use of a joystick-controlled piezoelectric micromanipulator with a glass needle attached to it to reposition chromosomes within dividing cells. A sample result shows the use of a micromanipulator to detach a chromosome from a spindle in a primary spermatocyte and to reposition that chromosome within the cell.

Figure 6 shows a sample micromanipulation of 2 adjacent grasshopper primary spermatocytes in several examples of the possible uses of micromanipulation. This experiment was done using an inverted, phase-contrast microscope. The 0:00 (times shown are in min:s) image shows both cells prior to the manipulation. One chromosome in the bottom cell is shown under tension applied by the micromanipulation needle (0:05; black arrow) and then completely detached from th...

Watch this Scientific Journal Video about Micromanipulation of Chromosomes in Insect Spermatocytes at JoVE.com

Micromanipulation of Chromosomes in Insect Spermatocytes

In this protocol, we describe the selection and preparation of appropriate cells for micromanipulation and the use of a piezoelectric micromanipulator to reposition chromosomes within those cells.

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and ...

This method can help answer key questions in cell biology, such as, how important is tension in satisfying the spindle checkpoint, and what is the role on spindle attachment in chromosome behavior in miosis? The main advantage of this technique is that it is a nonlethal technique that allows the experimenter to apply tension to, or reposition chromosomes in a living cell. To begin, prepare a 75 by 25 millimeter slide with a 20 millimeter diameter circular hole cut out of the center.Then, run a 25 by 25 millimeter number 1.5 cover slit through a bunsen burner flame for two seconds. Apply vacuum grease around the edge of the hole in the glass slide. Next, place the cover slip over the hole and press it to form a tight seal.Flip the slide over, and fill the newly created dissection well with halocarbon oil. After obtaining some adult male crickets, use dissecting scissors to cut through the dorsal surface parallel to the long axis of the abdomen, directly behind the wing buds. Then, gently squeeze the abdomen to push the testes through the cut in the exoskeleton.Using forceps, place the isolated testes into the dissection well. Under the dissecting microscope, use fine pointing forceps to divide the testes into smaller pieces, and remove any fat. Then, spread the contents of the testes under the oil on the surface of the cover slit.Continue to spread the contents of the testes until the spread portion is barely visible to the naked eye. Additional oil loaded dissection wells may be used if necessary. First, place the end of a glass tube in the flame of bunsen burner.Hold the end of the tubing to create a 150 degree angle and an extended area of narrow glass tubing. Break the tubing in the thin region so that the thin region extending from the previously created angle is approximately 10 millimeters long. Using the microforge, melt the tip of the glass needle, forming a 45 degree angle between the needle and the platinum wire of the microforge.Then, pull the glass away from the wire while simultaneously turning off the heat. Place the previously prepared slide onto the stage of an inverted phase contrast microscope. Then, find the dividing cells and center them in the field of view using the lowest magnification possible.Place the microneedle into the needle holder of the micromanipulator. Then, manually position the microneedle in the light path of the microscope. Focus the microscope several focal planes above the plane containing the cells.Next, use the joystick controller to reposition the microneedle several times to find the shadow of the needle in the x and y axes. Readjust the needle position until the position of the tip is visible, then, adjust the position of the needle along the z axis until the tip is in focus. Next, refocus on the cells, and focus the microscope just above the cell plane.Then, readjust the position of the needle so that the tip is in focus in this focal plane. Repeat this process using the higher magnification objective and a higher sensitivity setting. Refocus on the cells, and adjust their position, so they remain in the center of the field of view.Use the joystick to control the microneedle as it pushes chromosomes around inside the cell. Ensure the needle tip remains in the plane above the cells. To move the chromosome, focus on a chromosome near the top of the cell.Then, adjust the z axis on the joystick to bring the needle tip into focus, and move the needle with the joystick in x and y. Place the tip of the needle directly on the chromosome of interest, and push it in the desired direction. Apply enough tension to separate the chromosome from the spindle.Finally, place the chromosome anywhere within the cell. Using this protocol, it is possible to reposition, apply tension to, and completely detach a chromosome from a spindle using micromanipulation. In this example, two manipulated chromosomes were kept from reattaching to the spindle by being continually nudged with a micromanipulation needle.Once mastered, samples can be prepared for manipulation, and the micromanipulator can be positioned in 15 to 20 minutes. Micromanipulation experiments can take anywhere from a few seconds to several hours. While attempting this procedure, it's important to remember to manipulate chromosomes on the side of the cell farthest from the cover slip.After its development, this technique paved the way for researchers in the field of cell biology to explore the spindle checkpoint in grasshoppers and praying mantis.

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JoVE Journal

Developmental Biology

8.5K vistas.  Bucknell University. Este método puede ayudar a responder preguntas clave en la biología celular, como, ¿qué importancia es la tensión para satisfacer el punto de control del husillo y cuál es el papel en la fijación del husillo en el comportamiento cromosómica en la miosis? La principal ventaja de esta técnica es que es una técnica no letal que permite al experimentador aplicar tensión o reposicionar cromosomas en una célula viva. Para empezar, prepare un portaobjetos de 75 por 25 milímetros con un ...