產(chǎn)品規(guī)格:
    
        
            | 目錄號 | 產(chǎn)品名稱 | 規(guī)格 | 包裝 | 
        
            | 025 | Cell Counting Kit | 100次 | 1mL | 
        
            | 026 | Cell Counting Kit | 500次 | 5mL | 
        
            | 027 | Cell Counting Kit | 1000次 | 10mL | 
        
            | 028 | Cell Counting Kit | 3000次 | 30mL | 
    
產(chǎn)品簡介:
Cell Counting Kit簡稱CCK 試劑盒,為MTT法的替代方法,是一種基于WST(水溶性四唑鹽,化學(xué)名:2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑單鈉鹽)的廣泛應(yīng)用于細胞增殖和細胞毒性的快速高靈敏度檢測試劑盒。CCK法與其它檢測方法之間的比較:
    
        
            | 細胞計數(shù)方法 | MTT法 | XTT法 | WST-1法 | CCK法 | 
        
            | 形成的formazan的水溶性 | 差 | 好 | 好 | 好 | 
        
            | 產(chǎn)品性狀 | 粉末 | 2瓶溶液 | 溶液 | 1瓶溶液 | 
        
            | 使用方法 | 配成溶液后使用 | 現(xiàn)配現(xiàn)用 | 無需預(yù)制 | 無需預(yù)制 | 
        
            | 檢測靈敏度 | 高 | 很高 | 很高 | 高 | 
        
            | 檢測時間 | 較長 | 較短 | 較短 | 最短 | 
        
            | 檢測波長 | 560-600nm | 420-480nm | 420-480nm | 430-490nm | 
        
            | 細胞毒性 | 高,細胞形態(tài)完全消失 | 很低,細胞形態(tài)不變 | 很低,細胞形態(tài)不變 | 很低,細胞形態(tài)不變 | 
        
            | 試劑穩(wěn)定性 | 一般 | 較差 | 一般 | 很好 | 
        
            | 大批量樣品檢測 | 可以 | 非常適合 | 非常適合 | 非常適合 | 
        
            | 便捷程度 | 一般 | 便捷 | 便捷 | 非常便捷 | 
    
CCK用途:藥物篩選、細胞增殖測定、細胞毒性測定、腫瘤藥敏試驗
操作說明:
一、制作標(biāo)準(zhǔn)曲線(測定細胞具體數(shù)量時)
1、先用細胞計數(shù)板計數(shù)所制備的細胞懸液中的細胞數(shù)量,然后接種細胞。
2、按比例(例如:1/2比例)依次用培養(yǎng)基等比稀釋成一個細胞濃度梯度,一般要做3-5個細胞濃度梯度,每組3-6個復(fù)孔。
3、接種后培養(yǎng)2-4小時使細胞貼壁,然后加CCK試劑培養(yǎng)一定時間后測定OD值,制作出一條以細胞數(shù)量為橫坐標(biāo)(X軸),OD值為縱坐標(biāo)(Y軸)的標(biāo)準(zhǔn)曲線。根據(jù)此標(biāo)準(zhǔn)曲線可以測定出未知樣品的細胞數(shù)量(試用此標(biāo)準(zhǔn)曲線的前提是實驗的條件要一致,便于確定細胞的接種數(shù)量以及加入CCK后的培養(yǎng)時間。)
二、細胞活性檢測
1、在96孔板中接種細胞懸液(100μL/孔)。將培養(yǎng)板放在培養(yǎng)箱中預(yù)培養(yǎng)(在37℃,5% CO2的條件下)。
2、向每孔加入10 μL的CCK溶液(注意不要在孔中生成氣泡,它們會影響OD值的讀數(shù))。
3、將培養(yǎng)板在培養(yǎng)箱內(nèi)孵育1-4小時。
4、用酶標(biāo)儀測定在450nm處的吸光度。
5、如果暫時不測定OD值,打算以后測定的話,可以向每孔中加入10 μL 0.1M的HCL溶液或者1% w/v SDS溶液,并遮蓋培養(yǎng)板避光保存在室溫條件下。在24小時內(nèi)吸光度不會發(fā)生變化。
 
三、細胞增值-毒性檢測
1、在96孔板中配置100μL的細胞懸液。將培養(yǎng)板在培養(yǎng)箱預(yù)培養(yǎng)24小時(在37℃,5% CO2的條件下)。
2、向培養(yǎng)板加入10μL不同濃度的待測物質(zhì)。
3、將培養(yǎng)板在培養(yǎng)箱孵育一段適當(dāng)?shù)臅r間(例如:6、12、24或48小時)。
4、向每孔加入10μL CCK溶液(注意不要再孔中生成氣泡,它們會影響OD值的讀數(shù))。
5、將培養(yǎng)板在培養(yǎng)箱內(nèi)孵育1-4小時。
6、用酶標(biāo)儀測定在450nm處的吸光度。
7、如果暫時不測定OD值,打算以后測定的話,可以向每孔中加入10 μL 0.1M的HCL溶液或者1% w/v SDS溶液,并遮蓋培養(yǎng)板避光保存在室溫條件下。在24小時內(nèi)吸光度不會發(fā)生變化。
注意:如果待測物質(zhì)有氧化性或還原性的話,可在加CCK之前更換新鮮培養(yǎng)基(除去培養(yǎng)基,并用培養(yǎng)基洗滌細胞兩次,然后加入新的培養(yǎng)基),去掉藥物影響。當(dāng)然藥物影響比較小的情況下,可以不更換培養(yǎng)基,直接扣除培養(yǎng)基中加入藥物后的空白吸收即可。
 
備注:
①建議先做幾個孔摸索接種細胞的數(shù)量和加入CCK試劑后的培養(yǎng)時間。
②有條件的情況下建議采用多通道移液器,可以減少平行孔減的差異。加入CCK試劑時,建議斜貼著培養(yǎng)板壁加,不要插到培養(yǎng)基頁面下加,容易產(chǎn)生氣泡,會干擾OD值讀數(shù)。
③白細胞可能需要培養(yǎng)較長時間。
④當(dāng)使用標(biāo)準(zhǔn)96孔板時,貼壁細胞的最小接種量至少為1,000 個/孔 (100 μL 培養(yǎng)基)。檢測白細胞時的靈敏度相對較低,因此推薦接種量不低于2,500 個/孔 (100 μL 培養(yǎng)基)。如果要使用24孔板或6孔板實驗,請先計算每孔相應(yīng)的接種量,并按照每孔培養(yǎng)基總體積的10%加入CCK溶液。
⑤如果沒有450nm的濾光片,可以使用吸光度在430-490 nm之間的濾光片,但是450nm濾光片的檢測靈敏度最高。
⑥培養(yǎng)基中酚紅的吸光度可以在計算時,通過扣除空白孔中本底的吸光度而消去,因此不會對檢測造成影響。
 
活力計算:
細胞活力(%)=[A(加藥)-A(空白)]/[A(0加藥)-A(空白)] ×100
A(加藥):具有細胞、CCK溶液和藥物溶液的孔的吸光度
A(空白):具有培養(yǎng)基和CCK溶液而沒有細胞的孔的吸光度
A(0加藥):具有細胞、CCK溶液而沒有藥物溶液的孔的吸光度
細胞活力:細胞增殖活力或細胞毒性活力
保存條件:4℃干燥避光保存,有效期一年。
                         
                                             
                    
                        酶聯(lián)生物經(jīng)過不斷的實驗優(yōu)化和改進,積累了大量的經(jīng)驗,擁有專業(yè)的酶聯(lián)研發(fā)團隊。利用專業(yè)的酶聯(lián)免疫技術(shù)自主研發(fā)的elisa試劑盒,能對血清及其它樣本定量檢測抗原,定性檢測特異性抗體。優(yōu)質(zhì)的試劑,先進的儀器和正確的操作是保證ELISA檢測結(jié)果準(zhǔn)確可靠的必要條件。ELISA檢測的方便性、穩(wěn)定性、重復(fù)性和可靠性方面都具有很大的優(yōu)勢。
                        
                        ELISA檢測技術(shù)服務(wù)內(nèi)容: 
                            1、雙抗體夾心法檢測抗原 2、間接法檢測抗體 3、為客戶提供各種ELISA技術(shù)進行樣本檢測。
                            
                            以上代測費,凡購買本公司試劑盒,我們免費代測!
                            凡購買本公司目錄任何一種酶聯(lián)免疫檢測試劑盒,您只需將需要檢測的動物(Human, Rat, Mouse, Rabbit, Monkey,
                            Pig……)種類和檢測指標(biāo)(白介素類、激素類)及標(biāo)本數(shù)量(48T/96T)通知公司業(yè)務(wù)員即可。在接到客戶標(biāo)本當(dāng)日起,現(xiàn)貨產(chǎn)品一周內(nèi)將檢測報告交到客戶手中!
                            歡迎各科研單位在各種項目上與我們公司開展不同層次的密切合作,以雙贏求發(fā)展,共同進步,為中國檢測事業(yè)的發(fā)展積累經(jīng)驗。
                        
                        二、樣本要求
                            在收集標(biāo)本前都必須有一個完整的計劃,必須清楚要檢測的成份是否足夠穩(wěn)定。我們提倡新鮮標(biāo)本盡早檢測,對收集后當(dāng)天就進行檢測的標(biāo)本,及時儲存在4℃?zhèn)溆?,如有特殊原因需要周期收集?biāo)本,請造模取材后,將標(biāo)本及時分裝后放在-20℃或-70℃條件下保存。因冰室與室溫存在一定溫差,蛋白極易降解,直接影響實驗質(zhì)量,所以避免反復(fù)凍融。代測放免標(biāo)本的客戶取材前須向我司銷售人員索要說明書,具體操作注意事項請與我司技術(shù)人員溝通。
                        
                        液體類標(biāo)本:標(biāo)本必須為液體,不含沉淀。包括血清、血漿、尿液、胸腹水、腦脊液、細胞培養(yǎng)上清、組織勻漿等。
                        血清:室溫血液自然凝固10-20分鐘后,離心20分鐘左右(2000-3000轉(zhuǎn)/分)。收集上清。如有沉淀形成,應(yīng)再次離心。
                        血漿:應(yīng)根據(jù)試劑盒的要求選擇EDTA、檸檬酸鈉或肝素作為抗凝劑,加入10%(v/v)抗凝劑(0.1M檸檬酸鈉或1%heparin
                            或2.0%EDTA.Na2)混合10-20分鐘后,離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細收集上清。如有沉淀形成,應(yīng)再次離心。
                        尿液、胸腹水、腦脊液:用無菌管收集。離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細收集上清。如有沉淀形成,應(yīng)再次離心。
                        細胞培養(yǎng)上清:檢測分泌性的成份時,用無菌管收集。離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細收集上清。檢測細胞內(nèi)的成份時,用PBS(PH7.0-7.4)稀釋細胞懸液,細胞濃度達到100萬/ml左右。通過反復(fù)凍融,以使細胞破壞并放出細胞內(nèi)成份。離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細收集上清。保存過程中如有沉淀形成,應(yīng)再次離心。
                        
                        組織標(biāo)本:切割標(biāo)本后,稱取重量。加入一定量的PBS,緩沖液中可加入1μg/L蛋白酶抑制劑或50U/ml的Aprotinin(抑肽酶)。用手工或勻漿器將標(biāo)本勻漿充分。離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細收集上清置于-20度或-70度保存,如有必要,可以將樣品濃縮干燥。分裝后一份待檢測,其余冷凍備用。
                        
                        三、寄標(biāo)本時需注明以下情況:
                            1、標(biāo)本編號;2、所測項目;3、是否做復(fù)孔;3、聯(lián)系方式;4、實驗后標(biāo)本是否寄回。
                        客戶須知:
                            客戶應(yīng)對所提供的材料及信息負責(zé),如因客戶提供的材料及信息不準(zhǔn)確而引起的實驗延誤或經(jīng)濟損失由客戶承擔(dān)。
                     
                    
                                                
                            Q:1.
                                how to collect samples and preparation of ELISA? 
                            Performed by ELISA test is generally common clinical samples including blood (finger
                            blood, blood), urine, feces, cerebrospinal fluid, pleural effusion, prostatic fluid,
                            semen, vaginal secretions, which 
                            Some time of sample collection, preservation methods and has certain requirements.
                            
                            Collection (a) clinical specimens 
                            A, blood samples:Some physiological factors, such as smoking, eating, exercise, mood
                            swings, pregnancy, postural changes in blood can affect certain ingredients, even some
                            of diurnal variation. Therefore, blood samples 
                            Acquisition should avoid interference physiological factors, consistent with appropriate
                            conditions, such as can not be avoided, should indicate the factors on the specimen.
                            
                            1. Peripheral:Usually select the inside of blood left ring finger, the portion should be
                            no frostbite, inflammation, edema, damage. If the site does not meet the requirements to
                            other parts of the fingers instead. For burn patients, optional leather 
                            Intact skin at the blood. As part of routine blood tests (eg, white blood cell count,
                            sort, etc.) affected by physiological factors fluctuation is too large, when compared to
                            the conditional should be consistent. It relates to the body, blood clotting function
                            
                            Can test items (such as platelet count, bleeding time or clotting time) testing, we must
                            pay attention to understand whether the patient used anticoagulant, procoagulant drugs
                            in order to reduce or avoid interfering factors 
                            influences. 
                            2. Blood:In addition to involving a variety of projects such as hemostasis and
                            thrombosis detector requires the use of anticoagulated blood plasma, the current
                            analysis to detect the vast majority of projects can be directly detected using blood
                            serum. In the serum test items 
                            , Some (such as blood sugar, blood fat) diet and circadian factors influenced, fasting
                            blood samples were generally appropriate; some decay rapidly in the blood (serum enzyme
                            activity assay such as ACP activity, etc.), 
                            0 ~ 4 ℃ storage is not an activity decreased, the detection of these projects must be
                            timely and fast; some (such as creatine kinase) influenced by exercise and other
                            factors. Avoid hemolysis occurs when blood is also important 
                            And, more particularly potassium, LDH and other measurement. 
                            B, urine samples:With the same blood samples, urine samples affect diet, exercise,
                            medication and other factors that are also large, especially on the diet, so the morning
                            urine generally superior to random urine. Means getting up early morning urine 
                            After the first urine specimens, representing concentrated and acidified visible
                            components (such as blood cells, epithelial cells, tubular) easy to observe the relative
                            concentration. Random urine that is a random urine specimens convenient, but by diet,
                            
                            Sports, and even more the influence of drugs, prone to false positive and false negative
                            results, such as diet proteinuria, glucosuria diet, vitamin C interference occult blood
                            results and the like. Postprandial urine (patient 2 hours after lunch, collected 
                            Human Urine) suitable for urine, urine protein and urobilinogen check urine samples at
                            this time to increase the sensitivity of the test, the detection of minor lesions. 12
                            hours in urine cell count is Addis count (last night 8:00 
                            After emptying the bladder to all specimens of urine 8 o'clock the next morning),
                            because a long time, easy to breed bacteria shall be added preservative formaldehyde.
                            24-hour urine (the first day of the morning after emptying the bladder specimens from
                            8:00 to 8:00 the next morning 
                            All urine) quantification of chemical substances, including proteins, sugars, urinary
                            17-one, 17-hydroxy steroids, catecholamines, Ca2 +, etc., to detect different
                            substances, choose a different preservative preservative. clean 
                            Urine used for urine bacterial culture requires sterile specimens were taken after
                            washing the vulva. Urine specimens should be enough to collect all, at least 12 ml,
                            preferably 50 ml, the timing must collect all the urine of women 
                            Patients should avoid vaginal secretions, blood contamination of urine specimens. 
                            C, stool samples:Stool samples for the detection judgment digestive diseases has
                            important reference value. Collection requirements with a clean bamboo select faecal
                            mucus, pus and blood components and other abnormality, no abnormal appearance 
                            Droppings shall be drawn from multiple surface and deep manure end. Get parasitemia and
                            for egg counts should be collected 24 hours feces. Dysentery amoeba trophozoites check
                            should immediately check in after a bowel movement, and from there sepsis 
                            Softer at the drawn, insulation inspection. Charles S. japonicum eggs should take mucus,
                            pus and blood portion 30g stool specimens from at least miracidia hatching, and to be
                            treated as soon as possible. Check pinworm eggs must use transparent film swab 
                            Night before 12:00 or early in the morning from defecation wrinkled folds around the
                            anus and immediately swabbing at microscopic examination. Occult blood test (chemistry),
                            fasting before the test on the 3rd of meat and foods containing animal blood and ban
                            clothing iron, vitamin C and so on. 
                            Should be checked in all 1 hour stool specimen collection is completed, in order to
                            prevent damage to physical components of digestive enzymes and pH by. For clinical
                            samples above the detection indicators. 
                            D, CSF samples:CSF samples collected immediately after submission, place too long will
                            affect the test results: such as cell degeneration, destruction, leading to counting and
                            classification are not allowed; some chemicals such as glucose content will decompose
                            Save 
                            Less; bacteria occur autolysis affect bacteria detection rate. Cerebrospinal fluid
                            extracted three general dispensing a sterile tube, the first tube for bacterial culture,
                            a second tube for chemical analysis and immunological tests, the third tube for general
                            
                            Characters and microscopic examination, three of the order should be reversed. Specimen
                            collection is difficult because all inspection and testing process should pay attention
                            to safety. 
                            E, ascites and pleural effusion samples:CSF samples with the same attention to safety
                            after the specimen collection, and timely submission. Generally separated into three
                            tubes, one for routine cytology, a biochemical examination, a bacterial culture, in
                            order 
                            CSF same is appropriate. 
                            F, prostatic fluid sample:Prostatic fluid specimen after prostate massage by the
                            acquisition, directly drop when less liquid on a glass slide and timely submission shall
                            be taken to prevent sample evaporation to dryness, the amount collected for a long time
                            in a clean, dry test tube. If massage 
                            No prostatic fluid, urine sediment can be checked after the massage. 
                            G, semen samples:Abstinence before semen collection should be 3 to 7 days, drain the
                            urine after masturbation or other available methods of semen directly into clean
                            containers, insulation and timely submission. Due to changes in sperm production during
                            the day and 
                            Large, generally should be checked 2 to 3 times (each time interval of 1 to 2 weeks) in
                            order to make a diagnosis. 
                            H, samples of vaginal secretions:Vaginal samples were collected 24 hours before
                            intercourse should be prohibited, bath, vaginal examination, vaginal lavage and local on
                            the drug, etc., drawing instruments used need to be cleaned. Usually with brine-soaked
                            cotton swab from the vagina deep 
                            Or rear vaginal fornix, cervical canal mouth drawn, etc., made after saline smear
                            vaginal secretion samples observation, women with menstrual vaginal secretions were not
                            checking. 
                            2, do before each sample by ELISA experiment how to prepare? 
                            Before collecting the sample must have a comprehensive plan must clearly be detected
                            component is stable enough. To be collected on the same day
                            Sample testing, and timely backup stored at 4 ℃. For the next day re-testing samples
                            frozen in a timely manner after dispensing -20 ℃ spare, conditional, preferably -70 ℃
                            cryopreservation standby. Avoid repeated freezing and thawing specimens 
                            .
                            Liquid samples: including serum, plasma, urine, pleural effusion, cerebrospinal fluid,
                            cell culture supernatant and the like. 
                            1. serum: 
                            Coagulation at room temperature 10-20 mins, centrifugation 20 minutes or so (2000-3000
                            rev / min). Carefully collect the supernatant. If precipitation during storage,
                            Centrifugal again. 
                            2. Plasma: 
                            EDTA should be selected according to the requirements of the specimen, sodium citrate or
                            heparin as an anticoagulant, mix 10-20 mins, centrifugation 20 minutes or so (2000-3000
                            rev / min). Carefully collect the supernatant. Save process 
                            If precipitation appeared, Centrifugal again. 
                            3. Urine: 
                            Sterile collection tube. Centrifuged for 20 minutes or so (2000-3000 rev / min).
                            Carefully collect the supernatant. If precipitation during storage, Centrifugal again.
                            Pleural and peritoneal effusions, and cerebrospinal fluid Reference to this practice.
                            4. The cell culture supernatant: 
                            The detection of secretory component with a sterile collection tube. Centrifuged for 20
                            minutes or so (2000-3000 rev / min). Carefully collect the supernatant. 
                            5. cultured cells 
                            ????When the detection of intracellular components, diluted with PBS (PH7.2-7.4) cell
                            suspension, the cell concentration reached 1 million / ml or so. By repeated freezing
                            and thawing or tissue protein extraction reagent was added to the cells 
                            Damage and release of intracellular components. Centrifuged for 20 minutes or so
                            (2000-3000 rev / min). Carefully collect the supernatant. If precipitation during
                            storage, Centrifugal again. 
                            6. tissues 
                            ????After cutting samples, check the weight. Adding a certain amount of PBS, PH7.4.
                            Rapidly frozen with liquid nitrogen. After thawing samples remained at 2-8 ℃. Adding a
                            certain amount of PBS 
                            (PH7.4), or tissue protein extraction reagent, or by hand homogenizer homogenized
                            sample. Centrifuged for 20 minutes or so (2000-3000 rev / min). Carefully collect the
                            supernatant. A new package to be detected, which 
                            I alternate freezing.
                            
                            Q:Do
                                I have to run all of my standards and samples in duplicate?
                            
                            A:Yes, the duplicates are run in order to monitor assay precision and increase
                            confidence in the assay results obtained.
                            
                            Q:Do
                                I have to run all of my samples at one time?
                            
                            A:No, each kit uses stripwell microplate. This allows the user to analyse different
                            numbers of samples at different times.
                            
                            Q:What
                                types of reproducible results are obtained with the assays?
                            
                            A:Each kit comes with a manual containing a graph of typical data obtained. Any
                            variation in operator, pipetting and washing technique, incubation time or temperature,
                            and kit age can cause variation in result. Each user should obtain their own standard
                            curve.
                            
                            Q:Is
                                it possible to store the reagents other than indicated?
                            
                            A:Storage of the kit components under conditions other than indicated is not recommended
                            in order to assure proper performance of the test.
                            
                            Q:How
                                should I store my samples?
                            
                            A:Samples should be stored at -20oC or lower temperature. For long-term storage, it is
                            recommended to freeze them at -70oC -80oC.
                            
                            Q:Can
                                I modify the protocol?
                            
                            A:BG ELISA kits have been optimized to provide the best possible results. Modifying the
                            format or protocol may give inaccurate and wrong results.
                            
                            Q:Can
                                I use a sample type that is not recommended in the kit insert?
                            
                            A:The kit has been validated for the sample types listed in the kit insert. Sample types
                            other than those validated have not been tested. Contact Technical Service for further
                            information.
                            
                            Q:My
                                samples generated values that were outside the dynamic range of the assay. Can I use
                                these values?
                            
                            A:It is recommended that only sample values that fall within the range of the standard
                            curve be used. Values outside the range of the standard curve are generally non-linear,
                            which can lead to incorrectly extrapolated values. Samples that generate values higher
                            than the highest standard should be (further) diluted and the assay repeated. If samples
                            fall below the range of the assay, the sample is considered to be non-detectable.
                            
                            Q:Do
                                I have to run a Blank or Zero Standards every time?
                            
                            A:Yes, these are required for the calculations, and reflect any subtle but significant
                            performance changes from day to day and assay to assay. They are also extremely helpful
                            when troubleshooting the source of a particular assay problem.
                            
                            Q:Can
                                I alter the volume of sample I use in the assay?
                            
                            A:It is not recommended that you alter the volumes since all BG kits are designed for
                            optimal performance at the given volumes
                            
                            Q:Can
                                components from different kits be used?
                            
                            A:Each kit contains components which have specific lot numbers to ensure that all of the
                            components are performing optimally alone, as well as with all of the other components
                            in the kit. QC testing is performed on these specific lots. It is never recommended to
                            use your own components or components from other kits or vendors.
                            
                            Q:My
                                standard curve looked fine, but I didn’t get a signal in my sample when I expected
                                to, why?
                            
                            A:The sample may not contain the analyte. A matrix effect may be masking the detection.
                            Ensure that the recommended dilution was followed as stated in the kit insert. If
                            dilution was recommended, check to be sure that the dilution was performed properly.
                            Over-dilution may cause the sample to fall below the range of the standard curve.
                            
                            Q:How
                                do you recommend I wash my plate?
                            
                            A:If you are using an automated plate washer we recommend that the calibration be
                            checked on a regular basis, and that the system is flushed with the Plate Washing Buffer
                            prior to washing. The same is true for a manual washer. A repeater or a wash bottle can
                            also be used. The user should be careful to ensure that all of the contents are
                            aspirated and the plate tapped dry on lint-free paper.
                            
                            Q:Do
                                I need to use a plate shaker?
                            
                            A:Reliable results can be obtained without a plate shaker, but the O.D.'s will generally
                            be lower than those obtained using a plate shaker.
                            
                            Q:Why
                                do I have to use wavelength correction between 450-570nm?
                            
                            A:For the ELISA assay, reading at dual wavelengths is done to correct for the optical
                            density contributed by the plastic well, the lamp and optical fluctuations.
                            
                            Q:If
                                I extract my sample, do I still need to follow the recommended dilutions given in
                                the kit insert?
                            
                            A:The amount of sample dilution needed after an extraction procedure will be affected by
                            the effects of purification and concentration in the protocol used. The amount of
                            dilution or concentration will have to be determined by the end-user.
                            
                            Q:What
                                is the expected concentration of analyte that I should expect to find?
                            
                            A:The amount of a given analyte may vary not only from species-to-species, but also
                            between tissue and cellular sources. The best source of this information is the current
                            literature that is easily accessed through the Internet at multiple scientific
                            databases.
                            
                            Q:My
                                optical densities were a little higher (or lower) than those in the manual that came
                                with my kit. Why?
                            
                            A:The optical density is affected by a number of physical conditions such as time and
                            temperature. We suggest that you shorten or lengthen the final incubation with substrate
                            solution to compensate.
                            
                            Q:What
                                are the reasons for High Background?
                            
                            A:1) Improper Washing: Check volume of washing buffer reservoir and make sure all
                            recommended washing steps are performed.
                            2) Contaminated Substrate: Make sure there is no contamination of the substrate with
                            metal ions or oxidizing reagents, before use. Keep the extra substrate solution
                            separately during the ELISA substrate development time.
                            3) Substrate exposed to light: Exposure to light may result in a blue color of the
                            substrate. Keep solutions in the dark (vial) until ready to dispense into the plate.
                            4) Wrong Incubation Times/Temperatures: Generally follow the test protocol regarding
                            incubation times and temperatures. However, if all wells are intensely and equally
                            colored with no intensity gradient observed in the standard dilution series, then it may
                            be necessary to observe the substrate reaction as the color is developing, in order to
                            stop the reaction sooner.