This work was supported by the Neurotrauma Research Program (Western Australia). This project is funded through the Road Trauma Trust Account, but does not reflect views or recommendations of the Road Safety Council.

1.光学的較正：光出力を測定する<ol><li>光送達装置を調製するために、適切な電源に広帯域光源（ 例えば 、キセノン又はタングステンランプ）を接続する。光の平行ビームを生成する光源の前にコリメートレンズを配置します。光ビームからの熱の大部分を除去するために液体の熱フィルタを通して光を通過させる。用途に応じて、光のより柔軟な送達を提供する液体光ガイドの入口開口部上にコリメートされたビームの焦点を合わせる（ 例えば 、インキュベーター内に）。 </li><li>液体ライトガイドの他端側に、第2コリメートレンズと、干渉および中性密度フィルタ用のホルダーを配置する。この配置は必要な波長帯と強度の光の均等に照射スポットを生成することを確認してください。 注：光ガイドと試料面の端部との距離が依存して変化する必要があるかもしれ点灯しますが、ライトガイド増加の端からの距離が、光強度が減少することを覚えなければなりません領域。本研究では、この距離は14センチメートルで均等に96ウェルプレートに3×3のウェル領域を照明ビーム断面を生成する。 </li><li>ランプと関連する光学部品からの迷光が試料に到達できないことを確認してください。 </li><li>液体熱フィルタ用のウォータークーラーの電源を入れ、フィルターのジャケットを通る水の交換があることを確認。ランプ電源をオンにして、安定させるために広帯域光源のために少なくとも5分間待ちます。注：水冷却器の使用は、装置の過熱を防止するために必要とされる。 </li><li>照明の所望の波長帯を生成するために（通常、中心波長、ピーク透過率及び半値（FWHM）帯域幅全幅によって記述）狭帯域干渉フィルタを選択する。注：現在の研究で使用した狭帯域干渉フィルタは4であった42ナノメートル、550ナノメートル、671 nmおよび810 nmの。 </li><li>標本は、処理中に置かれるべき面でランプの光を測定します。製造業者の説明書に従って適正ソフトウェアを使用して、適切な分光放射計に接続された較正された放射照度プローブ（コサインコレクタ）を使用して光を測定する。 </li><li>所望の出力が得られるまでの光の強度を調整するために減光フィルタを使用する。注：本研究では、各干渉フィルタを通過した光の強度は、4つの治療波長帯の各々で同じ量子的出力を与えるように調整される。ランプ出力を変更することがありますようにそれは、定期的にキャリブレーションを確認することが重要です。 注：装置のセットアップ後、細胞を照射することができる状態にある図1現在の研究で使用される光伝送装置の図である。 </li></ol><img alt="図1"SRC = &quot;/ファイル/ ftp_upload / 52221 / 52221fig1.jpg&quot; /&gt; 光送達装置。イラストの図1の画像は、光動力源、ハウジング、コリメートレンズ、水フィルター、入口開口部は、液体ライトガイド、第2コリメートレンズ、フィルターホルダー、処理フレームとマットブラックカードとキセノンランプである。狭帯域波長および強度フィルタが示されていないことに注意してください。 2.細胞調製<ol><li>記載R / NIR-LT配信方法は、任意の細胞型またはin vitroモデル系で適用することができる。細胞培養のような以下の説明は、培養褐色細胞腫（PC12）、ミュラー（RMC1）及び一次混合網膜細胞への十分に確立された技術を用いて、一般的なものである。 </li><li>光線処理および酸化ストレスアッセイのための細胞播種の前に、培養物は、T75のトロボの活用の適切な補助剤（ 例えば 、FBS、抗生物質）を含有するそれぞれの増殖培地中で細胞を不死化70〜80％コンフルエントになるまでKS。 </li><li> 例えば 、試験される細胞型に適した方法を使用して、T75フラスコから不死化細胞を剥離。、トリプシン。 96ウェルアッセイプレートを、1時間のために10μg/ mlのポリ-L-リジンで被覆アッセイプレートのウェル（ 例えば 、PC12細胞）またはのための10μg/ mlのポリ-L-リジンでの細胞の播種に進む前に、必要に応じて10μgのとO / Nインキュベートし1H / mlのラミニン（ 例えば 、混合した網膜細胞）。 </li><li>遠心分離し、細胞のような適切な収集する（ 例えば 。、RMC1細胞用218×gでPC12細胞または10分間405×gで3分）、適切な細胞培養培地の8ミリリットルで上清と再懸濁を削除します。 </li><li>混合した網膜細胞を使用する場合は、確立された手順19に従って、酵素消化（パパイン）によるP0-5新生児ラットの子から準備</li><li>血球計を使用して、0.4％（w / v）のトリパンブルー色素を除いた生存細胞の数を数える。 </li><li>細胞密度のSUCを調整細胞は、培養中の24または48時間後に約70〜80％コンフルエントになる時間。 （PC12細胞のための播種密度はRMC1細胞については、2.5×10 5生存細胞/ mlで、混合網膜細胞を、8×10 5生存細胞/ mlで、4.0×10 5生存細胞/ mlである）。透明または黒色96ウェルプレート（それぞれH 2 O 2またはDCFH-DA分析のために使用される）のいずれかにセルを追加した後、適切な増殖培地100μlに、プレートを37℃で平坦な表面の上に座ってすることができ、5 ％CO 2細胞接着を可能にするために、少なくとも24時間（または任意の条件は、特定の細胞型に適している）。 </li><li>適切な96ウェルトレイ内の増殖培地で培養細胞彼らは70〜80％コンフルエント（PC12とRMC1細胞について24時間、混合網膜細胞のために48時間）になるまで。 </li></ol> 3.細胞にグルタミン酸ストレッサーを追加する<ol><li>適切なフルグローにおける0-10mMのL-グルタミン酸モノナトリウム塩の水和物ストレッサー濃度を準備第培地。 </li><li>細胞から培地を除去し、静かにPBS（PC12）またはHBSS（RMC1および混合網膜細胞）で細胞を3回洗浄する。洗浄後、100μl/ウェルの総容積にグルタミン酸含有培地を追加します。 </li></ol>光処理のまず投与量<ol><li>直ちにグルタミン酸または任意のストレッサーを添加すると、準備された光送達装置の下に配置することによって、所望の波長および強度の光治療に細胞をさらす。投与されている波長に応じて減光フィルタの確立組み合わせを使用して光ビームの量子的出力を変更してください。 注：現在の実験では、37°C ​​のヒートパッド96ウェルプレートを休止することによって温度を維持するために3分間光治療に細胞をさらす。必要に応じて処理時間を変化させてもよい。 <ol><li> 96 WEL内の隣接するウェルに光反射を防止するために、細胞の下にマットブラックカードを置きますLトレイは、他の治療パラメータに指定された。 </li></ol></li><li>治療は、時間のより長い長さのために所望される場合、細胞培養培地のpHを維持するか、理想的には、CO 2 CO 2 5％に調整濃度、または最適な条件下でインキュベーター内装置および処理プレートを配置し、25mMのHepes緩衝液を追加特定の細胞型のために。 </li><li>光処理の終了後、水平な面に細胞を置き、24時間37℃、5％CO 2（または細胞型のための最適なあらゆる条件）でインキュベートする。 注：上記のように必要に応じて、R / NIR-LTの追加の投与量は、投与することができる。 </li></ol> ROSの光処理と検出5.最終剤<ol><li>光治療の最終ラウンドを実行する前に、試薬はROSの検出に使用する準備。 50mMのクエン酸緩衝液（pH6.0）、トリトンX100、およびH 2 O 2検出試薬加工を準備溶液（1：2：10mMのH 2 O 2検出試薬原液の97比; 10 U / mlの西洋ワサビペルオキシダーゼ、50mMのクエン酸ナトリウム（pH6.0）中）。適切なメディアで100μMの最終濃度でDCFH-DAを準備します。 注：PC12細胞についてのDCFH-DA試薬RPMI培地中で、RMC1細胞のためのDCFH-DA試薬は、DMEM培地である。市販の検出試薬は、特定のROS差動感度を有しており、試薬は、個々の用途のために必要な情報を提供するように慎重に選択されるべきであることに留意されたい。 </li><li>ステップ4.1-4.1.2で説明したように、細胞に光治療を管理します。細胞が光の所望のフルエンス/波長で治療を受けていることを確認してください。 </li><li>直ちにグルタミン酸含有培地を除去し、適当な緩衝溶液で2回洗浄し、光処置後（PC12細胞のために、PBSを使用し、RMC1細胞について、HBSSを用いている）。次のように細胞上のROSアッセイを実行します。 <ol><li> H 2 O 2 </sub&gt;のアッセイ：各ウェルに50mMのクエン酸緩衝液（pH 6.0）の45μlを添加し、5μlのトリトンX100。穏やかに30秒間、オービタルシェーカー上で細胞を振って15分間CO 2を 37℃でインキュベートし、5％。 H 2 O 2の検出作業溶液50μlを加え、室温で30分間インキュベートする。 530nmの励起波長および480nmの発光波長でプレートリーダーを用いて蛍光を測定する。 </li><li> DCFアッセイ：各ウェルにDCFH-DAの100μM溶液100μlを添加し、37℃で30分間インキュベートし、5％CO 2。 100μMDCFH-DAを含む培地を除去し、（上記のように）を2回適当な緩衝液で洗浄する。各ウェルに緩衝液90μlのと10μlのトリトンX100を加え、穏やかに37℃で15分間のインキュベーションの前に30秒間のオービタルシェーカー上振る。 480nmでの励起波長および発光ヴァヴェルでプレートリーダーを使用してDCF由来の蛍光を測定する530nmののength。 </li><li>エクスプレスROSタンパク質1mgを計算するために標準曲線を用いて、製造者の指示に従ってタンパク質濃度を定量するための比色キットを使用して、ウェル内に残っている細胞のタンパク質濃度に相対値。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

我々が正常にR / NIR-LT インビトロの最適化の研究のためのメカニズムを提供するための正確かつ較正された送達システムを適応している。 R / NIR-LTの波長および強度のパラメータは、このシステムを使用して、正確かつ効率的に操作されることが可能である。我々は、ROSは、試験した細胞型において、送達波長および用量で減少しなかったが、細胞の光治療は、細胞死をもたらさなかっ...

活性酸素種（ROS）は、シグナル伝達経路および神経保護1のものを含む細胞代謝の正常な反応の範囲のために必要とされる。内因性の抗酸化機構がROSの産生を制御することができないしかし、細胞が酸化ストレス2,3に屈することができる。 CNSへの傷害後、ROS及び酸化ストレスの存在下での関連する増加は、損傷4,5の進行において重要な役割を果たしていると考えられている。評価されている酸化ストレスを減衰させるための戦略の大規模な数にもかかわらず、神経外傷6以下の臨床使用においてROS産生と関連した酸化ストレスを減衰させるには完全に有効な、臨床的に関連する抗酸化戦略は現在のところ存在しない。そのため酸化ストレスの減衰が治療的介入7のための重要な目標である。 改善が続くるR / NIR-LTは、傷害、おそらく酸化ストレスを減少させることによって噴門梗塞サイズ、糖尿病時の腎臓および肝臓の合併症、網膜変性、CNS損傷および脳卒中8の減少を含む、広範囲の疾患において報告されている。 CNS損傷に特に関連して、670nmの光の効果の前臨床試験は、網膜変性9-11、脊髄損傷12、ニューロン死13のモデルにおいて良好な効果を示した。臨床試験は、乾燥加齢黄斑変性症のために実施し、ストローク14のために、現在進行中でされてきた、しかし、これらの試験の結果はおそらく効果的な治療パラメータ15を採用する障害のため、有望な表示されません。このように、R / NIR-LTが広く、管理が容易な、非侵襲的で比較的安価な治療にもかかわらず、神経外傷の通常の臨床診療の一部として採用されていない。臨床翻訳への障壁は、CLの欠如、早期の標準化された効果的な治療プロトコル16,17の作用と不在の仕組みを理解していた。現在の光療法に関する文献は、照射源（LEDやレーザ）に対する治療パラメータの変動の過多を明らかにし、照射の波長（ 例えば 、630、670、780、810、830、880、904nm）、総投与量（ジュール/単位面積）、持続時間（露光時間）、タイミング（前または後の傷害）、配信の治療頻度及びモード（パルスまたは連続）8。研究間の治療パラメータの変動は、比較を困難にし、有効性16に関して懐疑的な見方に貢献してきました。 従って、R / NIR-LTの最適化が明らかに複数の変数を比較するために必要なハイスループットスクリーニング機構を提供することができる細胞培養系で、必要とされる。しかしWA上に十分な柔軟性と制御を提供することができ、いくつかの市販の照明システムがあるvelengthおよび強度は、このような最適化実験を行う。市販のLEDデバイスは、一般に、強度だけではなく変化し得る異なるメーカーからの複数のLED素子を用いた研究者らは、その結果、複数の波長または強度を、提供することができ、また、発光波長のスペクトルではない。我々は、狭帯域干渉フィルターを通して濾過し、広帯域キセノン光源を用いて光の波長およびフルエンスの範囲を生成し、R / NIR-LTのパラメータの近くに、正確な制御を可能にすることによって、この問題に対処している。 これは、処置の治療用量は、R / NIR-LTの場合、シトクロムcオキシダーゼ （COX）18であると仮定され、photoacceptor（発色団）と相互作用する光子の数によって定義されることに留意することが重要である。配信光子エネルギーは、波長に応じて変化する。異なる波長におけるエネルギーの等しい用量を意味するであろうコム光子の異なる数の珍重。したがって、装置から出射された光は、試験されるべき選択された波長の各々についての光子の数と同じ数を放出するように較正した。我々は、in vitroでの細胞への波長および強度の範囲でR / NIR-LTを送達するために用いることができるシステムを開発したが施さ細胞におけるROS産生に対する配信R / NIR-LTの効果を測定する能力を実証しているグルタミン酸ストレス。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OxiSelect Intracellular ROS Assay Kit (Green Fluorescence)</td>
			<td>Cell Biolabs</td>
			<td>STA-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed Reagent</td>
			<td>Molecular Probes</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>300 Watt Xenon Arc Lamp</td>
			<td>Newport Corporation</td>
			<td>6258</td>
			<td>Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on</td>
		</tr>
		<tr>
			<td>USB4000-FL Fluorescence Spectrometer</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CC-3-UV Cosine Corrector for Emission Collection</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#956;m diameter quartz fibre optic</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraSuite Spectroscopy Platform</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>2300 EnSpire Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Acute toxicity, wear gloves when handling.</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>142-47-2 (anhydrous)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pheochromocytoma rat adrenal medulla (PC12) cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI1640) Media</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
			<td>Gibco</td>
			<td>10082-147</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Horse Serum, New Zealand origin</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100X MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Retinal Muller (rMC1) cells</td>
			<td>University of California, San Diego</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>75cm2 Flasks</td>
			<td>BD Biosciences</td>
			<td>B4-BE-353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-134</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>1X Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>165U Papain</td>
			<td>Worthington</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 well plates, clear bottom, black</td>
			<td>Corning</td>
			<td>CLS3603-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile.</td>
			<td>Costar</td>
			<td>07-200-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Seesaw Rocker</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Density Filter Paper (0.3)</td>
			<td>THORLABS</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>442nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FL441.6-10</td>
			<td></td>
		</tr>
		<tr>
			<td>550nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB550-10</td>
			<td></td>
		</tr>
		<tr>
			<td>670nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB670-10</td>
			<td></td>
		</tr>
		<tr>
			<td>810nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB810-10e</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.1)</td>
			<td>THORLABS</td>
			<td>NE01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.2)</td>
			<td>THORLABS</td>
			<td>NE02B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.3)</td>
			<td>THORLABS</td>
			<td>NE03B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.5)</td>
			<td>THORLABS</td>
			<td>NE05B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.6)</td>
			<td>THORLABS</td>
			<td>NE06B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (1.0)</td>
			<td>THORLABS</td>
			<td>NE10B</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro

レーザーまたは発光ダイオード（LED）によって送達赤/近赤外光療法（R / NIR-LT）は、おそらく酸化ストレスを減少させることによって、in vivoでの中枢神経系損傷の範囲内の機能的および形態学的転帰を改善する。しかし、酸化的ストレスに対するR / NIR-LTの効果は波長や照射強度に応じて変化することが示されている。治療パラメータを比較した研究は、スループットインビトロ最適化研究において高適し複数の波長または強度を提供し、商業的に入手可能なデバイスが存在しないこと、に起因する欠けている。このプロトコルは、与えられた損傷モデルに必要な治療用量を最適化するための波長および強度の範囲で光を送達するための技術が記載されている。我々は、波長および強度のパラメータを容易に変更することができた光を送達する方法は、活性酸素種を減少させるためのR / NIR-LTの最適用量の決定を容易にすることができるという仮説を立てin vitroで（ROS）。 非コヒーレントキセノン光は、光の各波長（440、550、670及び810nmの中心波長）とフルエンス（8.5×10 -3 -1×10 3.8 J / cm 2程度） を送達するために狭帯域干渉フィルターを通して濾過し培養細胞へ。装置から出力された光は、各波長の光の治療に関連する、同じ量子的用量を放出するように較正した。グルタミン酸は、DCFH-DAとH 2 O 2感受性蛍光色素を使用して、光で処理した細胞を強調に反応種が検出された。 我々は成功し、CNS損傷のモデルとしてグルタミン酸に曝露された培養細胞に、生理的におよび治療に関連する波長および強度の範囲で光を配信。現在の研究で使用したR / NIR-LTのフルエンスは、培養細胞によって生成されたROSに影響を及ぼさなかったが、光送達の方法は、他のシステム経験にも適用可能であるemsのは、単離されたミトコンドリアまたはそれ以上の生理学的に関連する器官型スライス培養モデルを含む、酸化的代謝のアウトカム指標の範囲への影響を評価するために使用することができる。

670nmの波長で照射される光の出力は、以前に、生体内 （0.3 J / cm 2）を20 で有益であることが示さ670nmの光の量を包含するフルエンスの範囲で細胞を照射するために減光フィルタを用いて較正した。光源の前に減光フィルタの数が増加するにつれて、強度（W / m 2）が少ない光がターゲット領域に通過させる、減少した。表1は、波長フィルターを取り付けた光源か?...

Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

酸化ストレスに赤/近赤外光療法の波長および強度の範囲の効果の評価のための手法<em&gt;インビトロ</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

method-for-assessment-effects-range-wavelengths-intensities-rednear

Research

JoVE Journal

Biology

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

8.6K ビュー.  The University of Western Australia. Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

ビデオ: Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

光強度の変化にゼブラフィッシュの応答性を測定する行動アッセイ

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

生物学

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

グリコーゲンの生化学的滴定<em&gt;インビトロ</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

ゼブラフィッシュ胚における酸化ストレスの分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

の酸化ストレス抵抗を測定<em&gt;線虫（Caenorhabditis elegans）</em&gt; 96ウェルのマイクロタイタープレート中

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮細胞接着性の定量<em&gt;インビトロ</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸の濾過単離：簡易迅速DNA抽出方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP顕微鏡によるmiRNAのギャップジャンクション依存的転写の解析

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

マウスの腸内細菌叢に及ぼすバクテリオシンの影響を評価する方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

有害な効果の低圧プラズマ滅菌を使用して住んでいる携帯顕微鏡枯草菌胞子の生存に調査

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...