Thank you to Elena Mitsi (Research assistant) and Sr. Carole Hancock (Research nurse) and theatre staff for their contribution to filming, and to David Shaw (research nurse) for his support with our research bronchoscopies. Thanks also to our volunteer Rebecca Dunphy for allowing this procedure to be filmed.

1. The Subjects are Met on Arrival by the Respiratory Research Nurse
<ol>
	<li>The subject has been fasting for &#62;4 hr for solid food and 2 hr for clear fluids1.</li>
	<li>The procedure is performed as a day case with appropriate pre- and post-procedure checks (as per standard hospital policy).</li>
	<li>The subject wears a hospital gown.</li>
	<li>A small gauge cannula is inserted (ideally into the left hand) and remains in situ until the end of the post-procedure recovery period1.</li>
	<li>Preprocedure observations are taken (blood pressure, heart rate, oxygen saturations [sats]).</li>
	<li>The subject is transferred to the bronchoscopy suite.</li>
</ol>
2. The Subject is Prepared for the Procedure in the Suite by a Respiratory Research Clinician Experienced in Bronchoscopy
<ol>
	<li>Monitoring equipment is attached including; 3-lead ECG, pulse oximeter1,10 and sphygmomanometer. Anesthetic support is not needed including written informed consent.</li>
	<li>Oxygen may be delivered via nasal cannula at up to 4 L/min, to achieve sats &#62;90%1.</li>
	<li>The operator checks the bronchoscope, this includes ensuring effective suction by aspirating sterile normal saline.</li>
	<li>Topical anesthesia with lidocaine is achieved in the nasal passages (using Instillagel1) and the oral mucosa (using xylocaine)</li>
	<li>If sedation is appropriate and requested by the subject, intravenous midazolam is administered1. An appropriate reversal agent (flumazenil) is immediately available.</li>
	<li>Resuscitation equipment should be readily available1,10.</li>
</ol>
3. The Bronchoscope is Inserted and Positioned
<ol>
	<li>Intubation is usually via the nose. If this is not possible due to nasal polyps, inflamed turbinates, or any discomfort then the subject is intubated via the mouth (a mouth-guard is used to prevent damage to the scope or the subject's teeth).</li>
	<li>Topical anesthesia at the larynx is completed using 4% lidocaine, usually a total of 4-6 ml is used. Commonly this causes coughing on instillation.</li>
	<li>The vocal cords are passed, and further mucosal anesthesia using 2 ml aliquots of 2% lidocaine at the carina, at the division of the right lower lobe (RLL) and right middle lobe (RML) and at the RML entrance.</li>
	<li>The bronchoscope is positioned within the RML, ideally in the medial segment, in a position where it is distal enough to be in a secure position but not too distal so that the airway collapses when suction is applied ('Wink test' using the suction button).</li>
	<li>Good positioning is indicated during the wink test by a bronchoscope that can be fully maintained in position by the bronchoscopist and an airway that does not fully close immediately on gentle suction.</li>
</ol>
4. The BAL is Performed
<ol>
	<li>Four 60 ml syringes are prefilled with warmed normal saline - 60 ml, 50 ml, 50 ml, and 40 ml in successive syringes. When the subject and bronchoscopist are ready the first syringe of saline is instilled by the bronchoscopy assistant whilst the bronchoscopist maintains the position in the RML.</li>
	<li>Gentle hand suction is then performed by the assistant using the same port and 50 ml syringe.</li>
	<li>This procedure is then repeated a further 3x, with a maximal volume of 200 ml used in our specific technique.</li>
	<li>The retrieved BAL fluid is expelled gently into labelled containers already held on melting ice. Glass containers may be presiliconized in order to inhibit cell attachment and maximize cell return.</li>
	<li>The BAL fluid appears hazy against the light with surface soap bubbles formed by surfactants.</li>
	<li>The bronchoscope is slowly fully withdrawn.</li>
	<li>The BAL fluid is transported without delay on ice for immediate processing.</li>
</ol>
5. Subject Recovery
<ol>
	<li>The subject is recovered for 2-4 hr by the respiratory research nurse on a ward.</li>
	<li>During this time they are allowed to rest and then only eat and drink &#62;60 min1 after the procedure (when their swallow is assessed as safe), in order reduce the risk of aspiration.</li>
	<li>Post procedural observations are monitored and recorded. A clinical examination occurs prior to discharge.</li>
	<li>The subject is briefed predischarge about the common side effects (fever, mild right submammary chest discomfort, sore throat) and given a contact number in case significant side effects occur1.</li>
	<li>The subject is followed-up at 1-5 days post-procedure with a clinical contact. This may be either over the telephone or in person.</li>
</ol>
6. Cells are Isolated in the Laboratory &#8211; Perform on Melting Ice Where Possible
<ol>
	<li>The volume of BAL fluid is recorded and filtered through a double layer of sterile gauze swab to remove mucus plugs into prechilled, sterile 50 ml centrifuge tubes.</li>
	<li>Cells are pelleted by centrifugation at 500 x g at 4 &#176;C for 5-10 min and washed by vortexing in 50 ml cold normal saline. Centrifugation is repeated once.</li>
	<li>Cells are resuspended in culture medium (RPMI-1640 + 10% fetal bovine serum + 2 mM L-glutamine + penicillin [40 IU/ml], streptomycin [75 IU/ml], and amphotericin B [0.5 IU/ml]) and differential counting performed using an equal volume of trypan blue and a hemocytometer.</li>
	<li>The cell suspension is normalized to the required density (we use 1 x 106 cells/ml), introduced by pipetting into appropriate tissue culture plates, and incubated at 37 &#176;C for 3 hr to allow macrophage adherence. 1 ml of this cell suspension per well is appropriate for 24-well culture plates.</li>
	<li>For lymphocyte work, after 3 hr of incubation, the culture medium is gently pipetted up and down 3x, and collected. This medium contains lymphocytes and nonadherent macrophages and may be used to assess lymphocyte function; it may also be used in purification steps e.g. with CD14 magnetic beads to remove macrophages.</li>
	<li>For macrophage work, after 3 hr of incubation, the medium is carefully removed with minimal pipetting and replaced with fresh, warm medium without antibiotics (RPMI 1640 + 10% fetal bovine serum + 2 mM L-glutamine). The macrophages are adherent to the tissue culture plate (see Figure 1).</li>
</ol>

<ol>
	<li>BTS, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+British+Thoracic+Society+Bronchoscopy+Guideline+Committee+-+A+sub-Committee+of+the+Standards+of+Care+Committee+of+the+British+Thoracic+Society.">The British Thoracic Society Bronchoscopy Guideline Committee - A sub-Committee of the Standards of Care Committee of the British Thoracic Society.</a> Thorax. 56 (Suppl I), 1-21 (2001).</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=Technical+recommendations+and+guidelines+for+bronchoalveolar+lavage+(BAL)+report+of+the+European+Society+for+Pneumology+Task+Group.">Technical recommendations and guidelines for bronchoalveolar lavage (BAL) report of the European Society for Pneumology Task Group.</a> Eur. Respir. , 561-585 (1989).</li><li>Gordon, S. B., Irving, G. R., Lawson, R. A., Lee, M. E., Read, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+trafficking+and+killing+of+Streptococcus+pneumoniae+by+human+alveolar+macrophages+are+influenced+by+opsonins.">Intracellular trafficking and killing of Streptococcus pneumoniae by human alveolar macrophages are influenced by opsonins.</a> Infect. Immun. 68, 2286-2293 (2000).</li><li>Gordon, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+immunoglobulin+responses+to+Streptococcus+pneumoniae+are+altered+but+not+reduced+in+human+immunodeficiency+virus-infected+Malawian+adults.">Pulmonary immunoglobulin responses to Streptococcus pneumoniae are altered but not reduced in human immunodeficiency virus-infected Malawian adults.</a> J. Infect. Dis. 188, 666-670 (2003).</li><li>Fullerton, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domestic+smoke+exposure+is+associated+with+alveolar+macrophage+particulate+load.">Domestic smoke exposure is associated with alveolar macrophage particulate load.</a> Trop. Med. Int. Health. 14, 349-354 (2009).</li><li>Jambo, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoalveolar+CD4++T+cell+responses+to+respiratory+antigens+are+impaired+in+HIV-infected+adults.">Bronchoalveolar CD4+ T cell responses to respiratory antigens are impaired in HIV-infected adults.</a> Thorax. 66, 375-382 (2011).</li><li>Eagan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fluid+immunoglobulin+from+HIV-infected+subjects+has+impaired+opsonic+function+against+pneumococci.">Lung fluid immunoglobulin from HIV-infected subjects has impaired opsonic function against pneumococci.</a> Clin. Infect. Dis. 44, 1632-1638 (2007).</li><li>Rose, A. S., Knox, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoalveolar+lavage+as+a+research+tool.">Bronchoalveolar lavage as a research tool.</a> Sem. Respir. Crit. Care Med. 28, 561-573 (2007).</li><li>Wright, A. K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Nasal+Challenge+with+Streptococcus+pneumoniae+is+Immunising+in+the+Absence+of+Carriage.">Human Nasal Challenge with Streptococcus pneumoniae is Immunising in the Absence of Carriage.</a> PLoS Pathog. , (2012).</li><li>Bolliger, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERS/ATS+statement+on+interventional+pulmonology+European+Respiratory+Society/American+Thoracic+Society.">ERS/ATS statement on interventional pulmonology European Respiratory Society/American Thoracic Society.</a> Eur. Respir. J. 19, 356-373 (2002).</li><li>Ettensohn, D. B., Jankowski, M. J., Duncan, P. G., Lalor, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoalveolar+lavage+in+the+normal+volunteer+subject.+I.+Technical+aspects+and+intersubject+variability.">Bronchoalveolar lavage in the normal volunteer subject. I. Technical aspects and intersubject variability.</a> Chest. 94, 275-280 (1988).</li><li>Ettensohn, D. B., Jankowski, M. J., Redondo, A. A., Duncan, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoalveolar+lavage+in+the+normal+volunteer+subject.+2.+Safety+and+results+of+repeated+BAL,+and+use+in+the+assessment+of+intrasubject+variability.">Bronchoalveolar lavage in the normal volunteer subject. 2. Safety and results of repeated BAL, and use in the assessment of intrasubject variability.</a> Chest. 94, 281-285 (1988).</li><li>De Brauwer, E. I., Jacobs, J. A., Nieman, F., Bruggeman, C. A., Drent, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoalveolar+lavage+fluid+differential+cell+count.+How+many+cells+should+be+counted.">Bronchoalveolar lavage fluid differential cell count. How many cells should be counted.</a> Anal. Quant. Cytol. Histol. 24, 337-341 (2002).</li><li>Mtunthama, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malawians+permit+research+bronchoscopy+due+to+perceived+need+for+healthcare.">Malawians permit research bronchoscopy due to perceived need for healthcare.</a> J. Med. Ethics. 34, 303-307 (2008).</li><li>Pereira, W., Kovnat, D. M., Snider, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+cooperative+study+of+complications+following+flexible+fiberoptic+bronchoscopy.">A prospective cooperative study of complications following flexible fiberoptic bronchoscopy.</a> Chest. 73, 813-816 (1978).</li><li>Huang, Y. C., Bassett, M. A., Levin, D., Montilla, T., Ghio, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+phase+reaction+in+healthy+volunteers+after+bronchoscopy+with+lavage.">Acute phase reaction in healthy volunteers after bronchoscopy with lavage.</a> Chest. 129, 1565-1569 (2006).</li><li>Gritzfeld, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Human+Pneumococcal+Carriage.">Experimental Human Pneumococcal Carriage.</a> J. Vis. Exp. (72), (2013).</li></ol>

Andrea M. Collins is funded by The Bill and Melinda Gates Foundation, and no potential conflicting interests. She has received funds from Merck (MSD) and GSK to attend conferences and lecture fees. 50 research BALs performed independently.
Jamie Rylance is funded by the Wellcome Trust, and has no conflict of interest. He has received no other funds. 70 research BALs performed independently.
Daniel G. Wootton is funded by an NIHR PhD Fellowship, and has no conflicts of interest. 40 research BALs performed independently.
Angela D. Wright has no potential conflicting interests. Assisted with over 100 research BALs.
Adam K. A. Wright has no potential conflicting interests. He has processed over 50 BAL samples.
Duncan G. Fullerton has no potential conflicting interests. 100 research BALs performed independently.
Stephen B. Gordon has no potential conflicting interests. He has received funds from Merck (MSD) and Novartis to attend conferences. 400 research BALs performed independently.

In order to achieve good results the following are helpful:
<ol>
	<li>Good anesthetisation at the base of the tongue and posterior oropharynx. This is important because it improves the subject&#8217;s procedural tolerance and overall comfort.</li>
	<li>Either a pediatric (outer diameter 3.4-3.6 mm) or adult bronchoscope (outer diameter 4.8-5.9 mm) can be used.</li>
	<li>If there is any discomfort during nasal intubation, oral intubation is preferred. In general nasal intubation is either a straightforward technique or should be abandoned.</li>
	<li>After initial instillation of lidocaine to the oropharynx, the subject may prefer the bronchoscope to be withdrawn pending the onset of anesthesia, especially if the bronchoscope is inserted orally.</li>
	<li>Ensure at least 10 x 10&#160;ml syringes are prefilled with only 2-4 ml of lidocaine. Air should be left in the syringes allowing improved delivery of the anesthesia directly onto cords and endobronchial mucosa.</li>
	<li>Since coughing during the procedure significantly reduces BAL yield; using warmed saline and having good mucosal anesthesia are important to reduce this.</li>
	<li>The 50&#160;ml syringes must be prefilled with warmed saline. It is common for the first 50 ml of normal saline instilled to yield between 10-20 ml due to dead-space loss and the remainder running into the terminal airways. Further aliquots return a greater proportion of fluid.</li>
	<li>The operator should always check the bronchoscope prior to intubation, including ensuring effective suction by aspirating sterile normal saline. Loss of suction can commonly occur due to kinking of the tube&#160;or leaks around the connections with the bronchoscope or suction device.</li>
	<li>During manual suction, ideally the airway should not collapse. If suction recurrently collapses the airway, BAL returns are reduced to a valve mechanism, and local inflammation and bleeding is more likely: this in turn makes the airways more irritable and cough more likely. Choosing the most appropriate RML subsegmental branch and using low suction pressures are therefore both important.</li>
	<li>When air is constantly aspirated recheck the connection of the syringe to the bronchoscope and also the position of the bronchoscope in the distal airway. If this is a recurrent problem, post-procedure ensure that there are no holes in the bronchoscope itself.</li>
	<li>BAL from smokers is often darker due to the presence of intracellular particulates. Smokers may tolerate the bronchoscopy less well, and have increased cough.</li>
	<li>The preferable BAL volume yield is &#62;100 ml. A yield of &#62;150 ml is excellent. We have not experienced any yields &#62;170 ml. There is no linear relationship between the cell yield and BAL volume yield.</li>
	<li>Yields of &#60;100 ml are, in our experience, more likely to lead to side effects such as cough, chest pain and fever. We advise that subjects with yields of &#60;100 ml be recovered in the left lateral position, this may aid drainage of any remaining lung fluid.</li>
	<li>Clinical contact should be made with the subject within 1-5 days to ensure no side effects.</li>
	<li>Midazolam sedation is used for &#60;50% of subjects in the UK and &#60;5% in Malawi. Midazolam does not appear to directly improve the tolerability; some operators feel that sedation may sometimes be counterproductive in maintaining a coherent (and reassuring) dialogue with the subject.</li>
	<li>In our experience, there is a significant learning curve with this technique. For example coordination between doctor and nurse takes time and yields are often lower at the beginning of the learning process.</li>
	<li>In HIV positive subjects, we use antibiotics prophylactically prebronchoscopy.</li>
</ol>

Background
Fiberoptic bronchoscopy was developed for clinical practice; it is widely used both diagnostically and therapeutically1,2. Bronchoalveolar lavage (BAL) removes nonadherent cells and lung lining fluid from the mucosal surface; biopsy is used to sample mucosal and submucosal tissues. In research environments, BAL allows sampling of innate (lung macrophages3-5), cellular (B- and T- cells6), and humoral (immunoglobulin7) responses within the lung.
BAL is internationally accepted for research purposes8 and since 1999 the technique has been performed in &#62;1,000 subjects in the UK and Malawi by our group. We use this technique in studies of innate, cellular, and humoral immune responses to pneumococcal antigen including experimental human pneumococcal carriage9,17, biomass smoke5, HIV and vaccination, and adjunct treatment studies into recovery from pneumonia. Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid.
In a research context BAL utilizes a different technique from that practiced by respiratory and intensive care physicians (often termed bronchial wash, washings, lavage, or BAL) who are aiming to gain diagnostic or therapeutic benefit. The research technique is designed to harvest cells and preserve viability to facilitate the growth of cells in ex vivo culture. For these reasons the research technique uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage. Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory1.
Goal
The overall goal is that the procedure should be safe and effective. Subjects should not experience any physiological disturbance and operators should consistently collect in excess of 100 ml of BAL from subjects. After the procedure, subjects should experience minimal side effects.
Safety
Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria. This protocol includes a description of the potential risks and the steps taken to mitigate them.
Contraindications to research bronchoscopy may be expressed as absolute or relative, and are included in our study protocol as exclusion and inclusion criteria. Our subjects are all screened to ensure full health.
Absolute contraindications include unstable cervical spine, unresponsive hypoxia, unstable angina, bleeding diathesis, and malignant cardiac arrhythmia.
Relative contraindications, including those conditions associated with increased complication rates, include:
General: poorly cooperative subject1, any significant general medical problem e.g. epilepsy, previous poorly tolerated bronchoscopy, known adverse reactions to lidocaine or midazolam, pregnancy, poor nutrition.
Respiratory: hypoxia [saturations (sats) on air &#60;94%], hypercapnia, unstable asthma1, significantly impaired respiratory function1 (FEV1&#60; 1 L), pulmonary hypertension.
Cardiovascular: uremia, within 6 weeks of myocardial infarction1, superior vena cava obstruction.
Other: immunosuppression (our group regularly performs this procedure in HIV positive subjects).
See Table 1 &#8211; Risks associated with BAL for research.

<table border="1" cellpadding="0" cellspacing="0" style="width:966px;" width="966">
	<tbody>
		<tr>
			<td style="width:244px;height:25px;">
			Material
			</td>
			<td style="width:161px;height:25px;">
			Company
			</td>
			<td style="width:151px;height:25px;">
			Catalogue Number
			</td>
			<td style="width:410px;height:25px;">
			Comments
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Fibreoptic bronchoscope, light and suction source
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Surgical gown, sterile gloves
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Sphygnomanometer
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For continuous patient monitoring
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Pulse oximeter
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For continuous patient monitoring
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			3 lead ECG monitor
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For continuous patient monitoring
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Nasal oxygen delivery 2-4l/min
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Lidocaine 10% spray (Xylocaine)
			</td>
			<td style="width:161px;height:26px;">
			AstraZeneca
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For topical anaesthesia
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Lidocaine hydrochloride 2%
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For topical anaesthesia
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Lidocaine gel (Instillagel)
			</td>
			<td style="width:161px;height:26px;">
			Farco-Pharma, Germany
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Normal saline, sterile 200ml
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Warm to 30oC
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			10ml taper-end syringe (x10)
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For administration of lidocaine
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Intravenous cannula 18G
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For administration of midazolam
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Midazolam
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For sedation
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Flumazenil
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Reversal of benzodiazepine sedation (emergency use only)
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			60ml taper-end syringes (x4)
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For normal saline injection and BAL fluid withdrawal.
			May require connector to attach to the injection port of the bronchoscope
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Sterile gauze swab
			</td>
			<td style="width:161px;height:26px;">
			Vernaid, UK
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Sterile container for BAL fluid
			</td>
			<td style="width:161px;height:26px;">
			See text and comments
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			Siliconised glass bottles reduce macrophage adherence. Alternatively, 50ml centrifuge tubes may be used (total capacity 200ml)
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Sigmacote
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			SL2
			</td>
			<td style="width:410px;height:26px;">
			Only if siliconised glass bottles used. NB: Harmful and flammable. If used, follow precautions detailed in manufacturer&#8217;s MSDS
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Centrifuge
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;"></td>
			<td style="width:410px;height:26px;">
			At least 4x50ml tube capacity. Refrigeration to 4oC preferred.
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Shandon Cytospin centrifuge
			</td>
			<td style="width:161px;height:26px;">
			Thermo Scientific, UK
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For differential count only
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			RPMI 1640 medium with L-glutamine
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			R8758
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Fetal bovine serum
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			F6178
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Penicillin-Streptomycin
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			P4333
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Amphotericin B
			</td>
			<td style="width:161px;height:26px;">
			Sigma, UK
			</td>
			<td style="width:151px;height:26px;">
			A2942
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Tissue culture plates
			</td>
			<td style="width:161px;height:26px;">
			Greiner Bio-One, UK
			</td>
			<td style="width:151px;height:26px;">
			662160
			</td>
			<td style="width:410px;height:26px;">
			Alternative products available from other suppliers
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Glass microscope slides
			</td>
			<td style="width:161px;height:26px;">
			unspecified
			</td>
			<td style="width:151px;height:26px;">
			
			</td>
			<td style="width:410px;height:26px;">
			For differential count only
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Shandon cytofunnel
			</td>
			<td style="width:161px;height:26px;">
			Thermo Scientific, UK
			</td>
			<td style="width:151px;height:26px;">
			A78710003
			
			</td>
			<td style="width:410px;height:26px;">
			For differential count only
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Shandon cytoclip
			</td>
			<td style="width:161px;height:26px;">
			Thermo Scientific, UK
			</td>
			<td style="width:151px;height:26px;">
			59910052
			</td>
			<td style="width:410px;height:26px;">
			For differential count only
			</td>
		</tr>
		<tr>
			<td style="width:244px;height:26px;">
			Hemacolor staining set (fixative, red and blue reagents)
			</td>
			<td style="width:161px;height:26px;">
			Merck, Germany
			</td>
			<td style="width:151px;height:26px;">
			111661
			</td>
			<td style="width:410px;height:26px;">
			Use according to manufacturer&#8217;s instructions
			</td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage (bal) for research; obtaining adequate sample yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Good clinical results are a BAL volume of over 100 ml and a subject who has experienced at most, minor discomfort (see Table 1). This requires a relaxed and cooperative subject. The clinician must be confident, fully prepared (Materials), and understanding towards the volunteer.
Inter- and intra-subject variability in cellular yield (including differential counts) has been described. Differential cell counts from healthy subjects are typically &#62;90% macrophages with small numbers of neutrophils (&#60;2%) and lymphocytes (5-10%). BAL cells centrifuged onto microscope slides can be stained with a differential nuclear stain to obtain this information (Figure 1). In Figure 1, BAL cells have been fixed and stained using Hemacolor staining according to the manufacturer&#8217;s instructions. At least 300 cells should be counted to obtain reliable results for macrophages, neutrophils and lymphocytes; 500 cells for rarer cell types.
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hazard</td>
			<td>Risk</td>
			<td>Reduced in this protocol by:</td>
		</tr>
		<tr>
			<td colspan="3">Effect of bronchoscopy and BAL1,14</td>
		</tr>
		<tr>
			<td>Mild discomfort</td>
			<td>&#60;25%14,#</td>
			<td>Appropriate analgesia and sedation. Confident and caring approach to the subject.</td>
		</tr>
		<tr>
			<td>Epistaxis</td>
			<td>&#60;1%#</td>
			<td>Mild pressure for nasal intubation only. If subject discomfort or narrowed aperture - oral intubation instead.</td>
		</tr>
		<tr>
			<td>Endobronchial hemorrhage (hemoptysis)</td>
			<td>&#60;0.1%*15</td>
			<td>No biopsies taken. Careful control during bronchoscopy - avoiding respiratory mucosa.</td>
		</tr>
		<tr>
			<td>Nausea, vomiting and aspiration pneumonia</td>
			<td>&#60;0.02%14,#</td>
			<td>Fasting for solid food &#62;4 hr preprocedure1. Adequate topical anesthesia. Semirecumbent position during procedure. Careful post-procedure observation.</td>
		</tr>
		<tr>
			<td>Fever</td>
			<td>0.0114 - 1%#,16</td>
			<td>Relates to inflammation, and may minimized by maximal collection of BAL. Single lobe BAL only.#</td>
		</tr>
		<tr>
			<td>Sore nose/throat and hoarseness</td>
			<td>&#60;25%14,#</td>
			<td>Adequate topical anesthesia of the nose, throat and larynx (minimising coughing).</td>
		</tr>
		<tr>
			<td>Infection</td>
			<td>&#60;0.1% in HIV negative, 1% in HIV positive14,#</td>
			<td>Standard bronchoscope washing procedure1. Single lobe BAL only. # Early recognition of infection: clinical examination within 1 hr and clinical contact 1-5 days after bronchoscopy.</td>
		</tr>
		<tr>
			<td>Chest pain/ cough</td>
			<td>&#60;0.2%14,#</td>
			<td>Single lobe BAL only. Maximal BAL collection.#</td>
		</tr>
		<tr>
			<td colspan="3">Effects of drugs: lidocaine, midazolam</td>
		</tr>
		<tr>
			<td>Arrhythmia</td>
			<td>Very rare*</td>
			<td rowspan="3">Maximum of 5 mg/kg14 lidocaine used. Warmed normal saline instilled. # Pulse oximetry (sats &#62;90% with oxygen supplementation), cardiac monitoring throughout procedure. Benzodiazepine antagonist (flumazenil) immediately available.</td>
		</tr>
		<tr>
			<td>Disorientation / agitation</td>
			<td>&#60;0.1%&#8224;#</td>
		</tr>
		<tr>
			<td>Cardio respiratory depression</td>
			<td>Very rare*</td>
		</tr>
	</tbody>
</table>
* Not experienced in our group in over 1,500 research procedures.
&#8224; Idiosyncratic rather than dose-dependent reaction.
# Our group's experience in over 1,500 research procedures.
Table 1. Risks associated with BAL for research. This includes the hazard or risk and how this is reduced by using the described technique. Effects of the bronchoscopy/BAL and also the effects of any medicinal drugs used are included.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/4345/4345fig1highres.jpg" width="500" /> Figure 1. A Microscope Slide Showing BAL Macrophages and Occasional Neutrophils. BAL cells have been fixed in methanol and stained with Hemacolor red (eosin Y) and blue (azure B).

Watch this Scientific Journal Video about Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield at JoVE.com

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

bronchoalveolar-lavage-bal-for-research-obtaining-adequate-sample

Research

JoVE Journal

Medicine

72.9K Views.  National Institute for Health Research. We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

Video: Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future therapies. Severe lung injury with a reproducible deterioration of pulmonary gas exchange and hemodynamics can be induced in anesthetized pigs using repeated lung lavages with warmed 0.9% saline (50 ml/kg body weight). Including standard respiratory and hemodynamic monitoring with clinically applied devices in this model allows the evaluation of novel therapeutic strategies (drugs, modern ventilators, extracorporeal membrane oxygenators, ECMO), and bridges the gap between bench and bedside. Furthermore, induction of lung injury with lung lavages does not require the injection of pathogens/endotoxins that impact on measurements of pro- and anti-inflammatory cytokines. A disadvantage of the model is the high recruitability of atelectatic lung tissue. Standardization of the model helps to avoid pitfalls, to ensure comparability between experiments, and to reduce the number of animals needed.

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Repeated pulmonary lavages in anesthetized pigs induce lung injury resembling major aspects of human acute respiratory distress syndrome (ARDS). For this purpose the lungs are repeatedly lavaged with 0.9% saline at 37 &#176;C. The goal of the protocol is a reproducible mitigation of gas exchange and hemodynamics for research in ARDS.

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

Preparing Zanba-Stir-Fried Tiebangchui (TBC) Sample for Processing Technology Optimization

To begin a single factor experiment to compare Tiebangchui, or TBC slice thickness, for processing technology optimization, prepare five test groups each having 30 grams of TBC, but different slice thickness. Then weigh 90 grams of Zanba, which is three times as much as that of the TBC in each group. Set the temperature and the time of the automatic stir fry machine to 140 degrees Celsius and 40 minutes respectively.After the machine has heated up to the set temperature, add the weighed amounts of TBC and Zanba into the machine. Then using a high speed smashing machine, grind the Zanba stir-fried TBC into powder. Next, weigh two grams of Zanba stir-fried TBC powder in a conical flask.To the sample in the flask, add three milliliters of ammonia solution followed by 50 milliliters of a mixture of isopropyl alcohol and ethyl acetate, having a volume ratio of one is to one. After recording the weight of the conical flask with the contents, ultrasonicate the TBC sample for 30 minutes at 220 volts and 40 kilohertz maintaining a temperature below 25 degrees Celsius. Weigh the conical flask with the sample once again after the ultrasonic extraction.Make up for the lost weight by adding the isopropyl alcohol and ethyl acetate mixture mentioned previously, and filter the sample solution. Evaporate 25 milliliters of the filtrate to dryness using a rotary evaporator at 40 degrees Celsius. Then dissolve the residue in five milliliters of a solution of 0.05 hydrochloric acid in methanol.Filter the solution through a 0.2 micron syringe filter, and analyze it by performing high performance liquid chromatography, or HPLC. Calculate the contents of the MDA and DDAs in different processing products according to the standard curve. Calculate the comprehensive score based on the results via the critic method.Following the method demonstrated above, compare the amounts of Zanba, processing temperatures, and times for processing technology optimization. The results of the single factor tests revealed that the comprehensive scores of Zanba stir-fried TBC were highest when the sliced thickness is two centimeters, the amount of Zanba is three times, the processing temperature is 120 degrees Celsius, and the processing time is 60 minutes. The results revealed the range of slicing thickness, amount of Zanba, processing temperature, and processing time to be used for future experiments.

Non-Invasive Biomarker Discovery for Tear Proteomics Using LC-MS

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

Author Spotlight: Advancing Tear Fluid Analysis Using a Standardized Protocol for Proteomics Research 

This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...