p2x1 Receptor (IC50 = 68 nM); P2X2 Receptor (IC50 = 214 nM)

Na+/Ca2+ 交换器反向模式 (NCXREV) 被 PPADS 四钠 a（1-30 μM；10-50 分钟）以浓度和时间依赖性方式抑制 [2]。四钠 PPADS 对重组和其他天然 P2XR 有效。人类 P2XR 对 PPADS 四钠的敏感性因亚型而异；最敏感的亚型是 hP2X1、-2、-3、-5 和 -7R，而 hP2X4R 的 IC50 分别约为 1-3 和 ~30 μM [3]。

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在培养的系膜细胞中，PPADS以剂量依赖的方式抑制核苷酸，但FCS不刺激增殖。在抗Thy1模型中，PPADS在早期（第3天）特异性和剂量依赖性地减少了肾小球系膜细胞增殖以及系膜的表型激活和轻微的基质扩张，但在晚期（第8天）没有减少。虽然在系膜溶解程度、炎性细胞流入、蛋白尿或血压方面没有获得一致的效果，但PPADS治疗增加了抗Thy1大鼠的血清肌酐和尿素。在培养的MC和分离的肾小球中检测到P2Y受体表达（P2Y2和P2Y6），并在抗Thy1疾病期间表现出短暂的显著增加。 结论：这些数据强烈表明细胞外核苷酸在介导MC损伤后早期MC增殖中的体内作用[4]。

PPADS 四钠 a（15-60 mg/100g 体重 (BW)；腹腔注射；每 12 小时一次，持续 8 天）可抑制系膜增殖性肾炎体内系膜细胞 (MC) 增殖，而不影响非 MC 体内增殖 [4 ]。

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Na（+）/Ca（2+）交换器（NCX）的主要功能是从细胞质中取出1个Ca（2+）并引入3个Na（2+）。细胞质Na（+）浓度的增加诱导了NCX反向模式（NCX（REV）），有利于Ca（2+）内流。NCX（REV）可以被以下物质抑制：KB-R7943，一种阻断电压依赖性和储存操作的Ca（2+）通道的非特异性化合物；SEA0400似乎对NCX（REV）具有选择性，但很难获得，SN-6仅在心肌细胞中显示出疗效。我们发现，P2X受体拮抗剂PPADS在豚鼠气管肌细胞中起着NCX（REV）抑制剂的作用。在这些细胞中，我们通过用LiCl替代NaCl和NaHCO（3）来表征NCX（REV），从而使用fura 2-AM增加细胞内Ca（2+）浓度（[Ca（2+）]i）。我们每10分钟分析一次NCX（REV）的5次连续反应，发现它们之间没有差异。为了评估不同NCX（REV）阻断剂的作用，构建了KB-R7943（1、3.2和10μM）和SN-6（3.2、10和30μM）的浓度反应曲线，而PPADS的作用具有时间和浓度依赖性（1、3.2,10和30μM）。PPADS的效力和疗效与KB-R7943相似，而SN-6的效力最低。此外，对D600和硝苯地平敏感的KCl诱导的收缩被KB-R7943阻断，但不被PPADS阻断。KBR-7943（10μM）也显著降低了KCl诱导的心肌细胞[Ca（2+）]i增加。我们的研究结果表明，PPADS可以作为一种可靠的药理学工具来抑制NCX（REV），其优点是它比KB-R7943更具特异性，因为它不影响L型Ca（2+）通道。[1]

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外周损伤引起的神经性疼痛与局部炎症以及局部募集的巨噬细胞、雪旺氏细胞和神经胶质细胞中一氧化氮合酶（NOS）和炎性细胞因子的过表达有关。我们在坐骨神经慢性压迫损伤诱导的单神经病小鼠模型中研究了一氧化氮合酶（NOS）和细胞因子在中枢（脊髓和丘脑）和周围神经系统（神经和背根神经节）中的时间过程和定位。ATP被认为是内源性疼痛介质。因此，我们还评估了嘌呤能信号传导在疼痛超敏反应中的作用，使用P2受体拮抗剂磷酸吡哆醛-6-偶氮苯基-2'，4'-二磺酸（PPADS）对疼痛行为、NOS和细胞因子进行了评估。从损伤后第3天开始每天服用PPADS，剂量和时间依赖性地降低了触觉异常性疼痛和热痛觉过敏。PPADS（25mg/kg）完全逆转了伤害性超敏反应，同时降低了参与疼痛信号传导的外周（受伤的坐骨神经和L4-L6同侧背根神经节）和神经系统中枢台阶（L4-L6脊髓和丘脑）中NO/NOS系统和IL-1β的增加。IL-6仅在外周神经系统中过表达，PPADS延长给药可降低其在坐骨神经中的表达。总之，我们假设NO/NOS和IL-1β在这种神经病变模型中具有促痛感作用，嘌呤能拮抗剂通过抑制其过度活动来减轻疼痛超敏反应[3]。

电生理学[2]
在电压钳条件下，使用双电极放大器记录了cRNA注射卵母细胞的核苷酸诱发膜电流。当填充KCl（3M）时，细胞内微电极的电阻为1-2MΩ。用含有（mM）NaCl 110、KCl 2.5、HEPES 5、BaCl2 1.8、pH 7.4-7.5的细胞外溶液持续灌注卵母细胞（5 ml min-1）。所有记录均在室温（18°C）下在-60至-90 mV的保持电位下进行。电生理数据最初以3 kHz的频率进行滤波，以20 Hz的频率在连接到MP100WSW接口的计算机上捕获，并使用商业软件显示。

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酶联免疫吸附试验（ELISA）评价脊髓背侧IL-1β含量[3]
通过使用酶联免疫吸附试验对假手术、CCI和PPADS治疗的CCI动物的脊髓进行IL-1β蛋白的定量测定。如上所述，采集L4-L6脊髓切片，快速冷冻并储存在-80°C下。将样品在0.25ml含有蛋白酶抑制剂混合物的冰冷磷酸盐缓冲盐水中均质化并离心。上清液用于测量IL-1β水平。采用Lowry法测定颗粒中的总蛋白含量。 对于IL-1β的测量，使用小鼠IL-1β的CytoSet Elisa试剂盒。捕获抗体和二次生物素化抗体的浓度分别为1.25和0.125μg/ml。重组蛋白产生的标准曲线范围为15-1000pg/ml。 链霉抗生物素蛋白过氧化物酶和四甲基联苯胺用于显色。用2N H2SO4停止显色反应，并在450nm处读取光密度。

卵母细胞制备和P2X受体表达[2]
用Tricaine（0.2%，wt/vol）麻醉非洲爪蟾，并通过断头处死（根据机构规定）。卵巢的解剖和切除，以及去卵爪蟾卵母细胞的制备，在其他地方已有详细描述[King等人，1997]。去卵泡卵母细胞不具有天然的P1或P2受体，否则可能会使激动剂活性的分析复杂化[King等人，1996a，b]。此外，去卵泡卵母细胞在很大程度上缺乏胞外ATP酶活性，从而避免了P2受体拮抗剂抑制胞外酶的复杂问题[Ziganshin等人，1995]。用编码大鼠P2X1或大鼠P2X3受体亚基的封端核糖核酸（cRNA，1mg/ml）在细胞质中注射（40nl）成熟卵母细胞（V期和VI期）。将注射的卵母细胞在18°C下在含有（mM）NaCl 110、KCl 1、NaHCO3 2.4、Tris-HCl 7.5、Ca（NO3）2 0.33、CaCl2 0.41、MgSO4 0.82的沐浴液（pH 7.5）中孵育48小时，并补充50μg/l的硫酸庆大霉素，以使受体完全表达，然后在4°C下储存长达12天。

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通过细胞计数和[3H]胸苷掺入试验测量PPADS对核苷酸或胎牛血清（FCS）刺激的培养MC增殖的影响。在诱导抗Thy1模型后，大鼠接受不同剂量（15、30、60mg/kg BW）的P2受体拮抗剂PPADS注射。在抗Thy1疾病期间评估增殖系膜和非系膜细胞、系膜细胞活化、基质积聚、炎性细胞流入、系膜溶解、微动脉瘤形成和肾功能参数。使用RT-PCR和实时PCR、Northern印迹分析、原位杂交和免疫组织化学评估P2Y mRNA和蛋白质表达[4]。

Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rats, body weight 160 to 200 g (anti-Thy1 disease model) [4]
Doses: 15 mg/100g BW, 30 mg/100g BW, 60 mg/100g BW
Route of Administration: intraperitoneal (ip) injection; every 12 hrs (hrs (hours)) for 8 days (The first PPADS injection was given 60 minutes after disease induction, and the loading dose always contained double the amount of PPADS compared with subsequent injections.)
Experimental Results: Early (day 3) specifically And dose-dependently diminished mesangial cell proliferation without changing non-MC proliferation.

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Drug treatment [3]
Pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid tetrasodium salt (PPADS) was dissolved in saline and used at doses of 6.25, 12.5 and 25mg/kg (0.1ml/10g). Doses were chosen according to those employed by Gourine et al. (2005) in order to attenuate fever and cytokine responses induced by lipopolysaccharide in rats. PPADS or saline was administered i.p. to neuropathic and sham-operated mice once a day for 11 days, starting from the third day after surgery. The effect of the acute administration of PPADS at the highest dose (25mg/kg) has been evaluated at both third and 14th day after lesion: behavioural evaluations were performed both 1 and 24h after administration. The same experimental protocol was applied in mice treated for 10 days with saline, i.e. 14 days after sciatic nerve ligation.

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Thermal hyperalgesia and mechanical allodynia [3]
Responses to thermal and mechanical stimuli were measured before and 3, 7 and 14 days (24h after the last administration with PPADS or saline) after the surgical procedure. Measurements were performed on both the ipsilateral and contralateral hind paws of all mice by researchers who were blind to treatments. Thermal hyperalgesia was tested according to the Hargreaves procedure (Hargreaves et al., 1988), slightly modified by us for mouse, using a Plantar test apparatus. Briefly, mice were placed in smaller clear plexiglass cubicles and allowed to acclimatize. A constant intensity radiant heat source (beam diameter 0.5cm and intensity 20 I.R.) was aimed at the midplantar area of the hind paw. The time, in seconds (s), from initial heat source activation until paw withdrawal was recorded. Mechanical allodynia was assessed using the Dynamic Plantar Aesthesiometer. Animals were placed in a test cage with a wire mesh floor, and the rigid tip of a von Frey filament (punctate stimulus) was applied to the skin of the midplantar area of the hind paw. The filament exerted an increasing force, ranging up to 5g in 20s, starting below the threshold of detection and increasing until the animal removed its paw. Withdrawal threshold was expressed in grams. Withdrawal threshold of ipsilateral and contralateral paws was measured four times and the value was the mean of the four evaluations.

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Biochemical evaluations [3]
The biochemical evaluations were performed on animals receiving the highest dose of PPADS (25mg/kg) always by researchers who were blind to treatments. At 3, 7 and 14 days following surgery, 24h after the last dose of saline or PPADS, nociceptive and mechanical thresholds were recorded. Immediately after behavioural evaluations, mice were anaesthetized with sodium pentobarbital (60mg/kg, i.p., 0.1ml/10g) and under dissecting microscope the ipsilateral sciatic nerve, proximal to the trifurcation (about 1cm), before the three ligatures in the CCI animals, the ipsilateral L4, L5 and L6 DRG, the lumbar dorsal spinal cord at L4–L6 level, and ipsilateral and contralateral thalamus were removed and immediately frozen in liquid nitrogen and stored at −80°C until the NOSs content and cytokine expression assay. In some experiments a small portion of ipsilateral sciatic nerve, proximal to the trifurcation, before three ligatures in the CCI animals, and lumbar spinal dorsal at L4–L6 level was used to prepare nuclear extracts, which were stored at −80°C until the transcription factor NF-κB was assayed. In other experiments, a small portion of sciatic nerve, between the ligatures in CCI animals and trifurcation, was stored at −80°C until the assay of myelin proteins. In view of the technical difficulty of measurement of NO, which requires accuracy in the time of sampling and prompt measurement immediately after sampling, due to the instability of NO and nitrite, we evaluated the level of NOSs (inducible and neuronal) to represent NO changes, as previously reported by Salake et al. (2000) and Wang et al. (2004).

[1]. PPADS, a P2X receptor antagonist, as a novel inhibitor of the reverse mode of the Na⁺/Ca²⁺ exchanger in guinea pig airway smooth muscle. Eur J Pharmacol. 2012 Jan 15;674(2-3):439-44.

[2]. Mapping the binding site of the P2X receptor antagonist PPADS reveals the importance of orthosteric site charge and the cysteine-rich head region. J Biol Chem. 2018 Aug 17;293(33):12820-12831.

[3]. The purinergic antagonist PPADS reduces pain related behaviours and interleukin-1 beta, interleukin-6, iNOS and nNOS overproduction in central and peripheral nervous system after peripheral neuropathy in mice. Pain. 2008 Jul;137(1):81-95.

[4]. P2 receptor antagonist PPADS inhibits mesangial cell proliferation in experimental mesangialproliferative glomerulonephritis. Kidney Int. 2002 Nov;62(5):1659-71.

[5]. PPADS is a reversible competitive antagonist of the NAADP receptor. Cell Calcium. 2007 Jun;41(6):505-11.

[6]. Actions of a Series of PPADS Analogs at P2X1 and P2X3 Receptors. Drug Dev Res. 2001 Aug;53(4):281-291.

[7]. Einfluss von ATP und seinen Derivaten auf die Aktivierung von Monozyten.

Tetrasodium 5'-phosphonatopyridoxal-6-azobenzene-2,4-disulfonate is an organic sodium salt that is the tetrasodium salt of 5'-phosphopyridoxal-6-azobenzene-2,4-disulfonic acid It has a role as a purinergic receptor P2X antagonist. It is an organic sodium salt and an organosulfonate salt. It contains a 5'-phosphonatopyridoxal-6-azobenzene-2,4-disulfonate.

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5'-phosphopyridoxal-6-azobenzene-2,4-disulfonic acid is an arenesulfonic acid that is pyridoxal 5'-phosphate carrying an additional 2,4-disulfophenylazo substituent at position 6. It has a role as a purinergic receptor P2X antagonist. It is an arenesulfonic acid, a member of azobenzenes, a member of methylpyridines, a monohydroxypyridine, a pyridinecarbaldehyde and an organic phosphate. It is functionally related to a pyridoxal 5'-phosphate. It is a conjugate acid of a 5'-phosphonatopyridoxal-6-azobenzene-2,4-disulfonate.

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Platelet Aggregation Inhibitors: Drugs or agents which antagonize or impair any mechanism leading to blood platelet aggregation, whether during the phases of activation and shape change or following the dense-granule release reaction and stimulation of the prostaglandin-thromboxane system.

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ATP is the native agonist for cell-surface ligand-gated P2X receptor (P2XR) cation channels. The seven mammalian subunits (P2X1-7) form homo- and heterotrimeric P2XRs having significant physiological and pathophysiological roles. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) is an effective antagonist at most mammalian P2XRs. Lys-249 in the extracellular domain of P2XR has previously been shown to contribute to PPADS action. To map this antagonist site, we generated human P2X1R cysteine substitutions within a circle centered at Lys-249 (with a radius of 13 Å equal to the length of PPADS). We hypothesized that cysteine substitutions of residues involved in PPADS binding would (i) reduce cysteine accessibility (measured by MTSEA-biotinylation), (ii) exhibit altered PPADS affinity, and (iii) quench the fluorescence of cysteine residues modified with MTS-TAMRA. Of the 26 residues tested, these criteria were met by only four (Lys-70, Asp-170, Lys-190, and Lys-249), defining the antagonist site, validating molecular docking results, and thereby providing the first experimentally supported model of PPADS binding. This binding site overlapped with the ATP-binding site, indicating that PPADS sterically blocks agonist access. Moreover, PPADS induced a conformational change at the cysteine-rich head (CRH) region adjacent to the orthosteric ATP-binding pocket. The importance of this movement was confirmed by demonstrating that substitution introducing positive charge present in the CRH of the hP2X1R causes PPADS sensitivity at the normally insensitive rat P2X4R. This study provides a template for developing P2XR subtype selectivity based on the differences among the mammalian subunits around the orthosteric P2XR-binding site and the CRH.[2]

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Although extracellular nucleotides have been shown to confer mitogenic effects in cultured rat mesangial cells through activation of purinergic P2 receptors (P2Y receptors), thus far the in vivo relevance of these findings is unclear. Virtually all cells and in particular the dense granules of platelets contain high levels of nucleotides that are released upon cell injury or platelet aggregation. In experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), mesangiolysis and glomerular platelet aggregation are followed by a pronounced mesangial cell (MC) proliferative response leading to glomerular hypercellularity. Therefore, we examined the role of extracellular nucleotides and their corresponding receptors in nucleotide-stimulated cultured mesangial cells and in inflammatory glomerular disease using the P2 receptor antagonist PPADS.[4]

C14H10N3O12PS2-4.4[NA+]

599.3051

598.903

C, 28.06; H, 1.68; N, 7.01; Na, 15.34; O, 32.04; P, 5.17; S, 10.70

192575-19-2

Iso-PPADS tetrasodium;207572-67-6

135484645

Orange to red solid powder

3.779

288.3

1

15

5

36

918

0

KURWUCJJNVPCHT-UHFFFAOYSA-J

InChI=1S/C14H14N3O12PS2.4Na/c1-7-13(19)9(5-18)10(6-29-30(20,21)22)14(15-7)17-16-11-3-2-8(31(23,24)25)4-12(11)32(26,27)28;;;;/h2-5,19H,6H2,1H3,(H2,20,21,22)(H,23,24,25)(H,26,27,28);;;;/q;4*+1/p-4

tetrasodium;4-[[4-formyl-5-hydroxy-6-methyl-3-(phosphonatooxymethyl)pyridin-2-yl]diazenyl]benzene-1,3-disulfonate

PPADS TETRASODIUM SALT; 192575-19-2; PPADS Tetrasodium; CHEMBL1256743; Pyridoxalphosphate-6-azophenyl-2',4'- disulfonic acid tetrasodium salt; 4-[[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid tetrasodium salt; PPADS tetrasodium salt, anhydrous; 4-[2-[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]diazenyl]-1,3-benzenedisulfonic Acid Tetrasodium Salt;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~83.43 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。