| Creatinine (CRE) is a metabolite produced by muscle metabolism, which is mainly excreted via glomerular filtration. Under normal physiological conditions, the internal creatinine level remains relatively stable. Serum creatinine concentration serves as one of the key indicators for evaluating glomerular filtration function. This assay kit adopts an enzymatic method: creatinine is specifically catalyzed by creatininase to generate creatine. Subsequently, creatine is sequentially acted upon by creatinase and sarcosine oxidase to produce hydrogen peroxide. Hydrogen peroxide reacts with chromogenic reagent to form purple-colored products with a maximum absorption peak at 546 nm, and the creatinine content is calculated accordingly. Components: | C1515993 | Components | Appearance | 48T | Storage | Quantity Per Test | | C1515993A | Reagent 1 | Yellow transparent liquid | 17.5 mL | 2-8℃. Store in the dark | 350 uL for 1 reaction | | C1515993B | Reagent 2 | Pink transparent liquid | 6 mL | 2-8℃. Store in the dark | 120 uL for 1 reaction | | C1515993C | Standard Sample | White powder | 2 mg | 2-8℃. Store in the dark | Use as per requirements |
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Standard sample preparation:
1. Before use, centrifuge 8000g at 4℃ for 2 minutes to ensure the reagents fall to the bottom of the tube;
2. Add 1 mL of distilled water to dissolve it to obtain a standard solution with a concentration of 2 mg/mL;
3. Dilute it 40 times with distilled water (20 μl of the standard solution + 780 μl of water) to obtain a 0.05 mg/mL solution, which is 442 μmol/L of creatinine standard solution for the test sample.
Experimental Instruments:
Mortar/homogenizer, analytical balance, ice box/ice maker, tabletop centrifuge, adjustable pipette, water bath incubator/drying oven/cell incubator/metal bath, 96-well plate, centrifuge tubes, microplate reader, distilled water (deionized water or ultrapure water is acceptable).
Index Determination:
It is recommended to select 1-3 representative samples with significant differences for pre-experiment to familiarize with operating procedures, and adjust sample concentration properly to avoid unnecessary waste of samples and reagents.
1. Sample Extraction
① Tissue samples: Weigh about 0.1 g tissue sample, add 1 mL normal saline or PBS buffer for homogenization. Transfer all crude extract into EP tube, centrifuge at 12000 rpm at room temperature for 10 min, and collect supernatant for detection.
② Liquid samples: Clear liquid samples can be detected directly; turbid samples need centrifugation before taking supernatant for assay.
③ Bacterial & cell samples: Collect bacteria or cells into centrifuge tubes and discard supernatant after centrifugation. Take approximately 5 million bacteria/cells and resuspend in 1 mL normal saline or PBS. Perform ultrasonic disruption in ice bath (power: 200 W, ultrasonic for 3 s, interval 10 s, repeat 30 times). Centrifuge at 12000 rpm, 4 °C for 10 min, collect supernatant and place on ice for later use.
Note: For increased sample dosage, extract samples at the ratio of cell number (10⁴ cells) : absolute ethanol (mL) = 500~1000 : 1.
Cell lysis buffer can also be used for sample treatment following the official protocol.
2. Detection Procedures
① Turn on the microplate reader, set temperature at 37 °C (skip temperature setting if unavailable), and adjust detection wavelength to 546 nm.
② Select 2 samples to confirm the optimal dilution factor (D) before formal detection.
③ Thaw all reagents to room temperature, and add reagents into 96-well plate in sequence.
| Reagent components (μL) | Testing tube | Blank tube (single run) | Standard tube (single run) | | Sample | 30 |
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| | Distilled water | 200 | 230 | 200 | | Standard sample |
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| 30 | | Reagent 1 | 350 | 350 | 350 | | Mix well, incubate at 37℃ for 5 minutes, and read the absorbance value A1 at 546nm. | | Reagent 2 | 120 | 120 | 120 | | Mix well, incubate at 37℃ for 5 minutes, then read the absorbance value A2 at 546nm. ΔA = A2 - A1. |
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Notes:
1.If the ΔA value of test well exceeds 0.5, dilute the sample with distilled water and bring dilution factor D into calculation formula.
2.If ΔA is lower than 0.005, increase sample loading volume V1 (e.g. from 6 μL to 10 μL or more, reduce Reagent 2 volume correspondingly; blank and standard wells shall be adjusted in the same way) or increase sample weight W. Recalculate results with revised parameters.
Result Calculation:
1. Calculated by sample weight
Creatinine content (nmol/g) = (Cₛₜₐₙdₐᵣd × V₂) × (ΔAₜₑₛₜ - ΔAbₗₐₙₖ) ÷ (ΔAₛₜₐₙdₐᵣd - Δ Abₗₐₙₖ ) ÷ ( V₁ ÷ V × W) × D = 442 × (Δ Aₜₑₛₜ - ΔAbₗₐₙₖ) ÷ (ΔAₛₜₐₙdₐᵣd - ΔAbₗₐₙₖ) ÷ W × D
2. Calculated by protein concentration
Creatinine content (nmol/mg prot) = (Cₛₜₐₙdₐᵣd × V₂) × (ΔAₜₑₛₜ - ΔAbₗₐₙₖ) ÷ (ΔAₛₜₐₙdₐᵣd - Δ Abₗₐₙₖ) ÷ (V₁ ÷ V × Cₚᵣ× D = 442 × (Δ Aₜₑₛₜ - ΔAbₗₐₙₖ) ÷ (ΔAₛₜₐₙdₐᵣd - ΔAbₗₐₙₖ) ÷ Cₚᵣ× D
3. Calculated by volume
Creatinine content (μmol/L) = ( Cₛₜₐₙdₐᵣd × V₂ ) × ( ΔAₜₑₛₜ - ΔAbₗₐₙₖ ) ÷ ( ΔAₛₜₐₙdₐᵣd - Δ Abₗₐₙₖ ) ÷ V₁ × D = 442 × ( Δ Aₜₑₛₜ - ΔAbₗₐₙₖ ) ÷ ( ΔAₛₜₐₙdₐᵣd - ΔAbₗₐₙₖ ) × D
4. Calculated by cell number
Creatinine content (μmol/10⁴ cells) = (Cₛₜₐₙdₐᵣd × V₂) × ( ΔAₜₑₛₜ - ΔAbₗₐₙₖ ) ÷ ( ΔAₛₜₐₙdₐᵣd - Δ Abₗₐₙₖ ) ÷ (500 × V₁ ÷ V) × D = 0.884 × ( Δ Aₜₑₛₜ - ΔAbₗₐₙₖ ) ÷ ( ΔAₛₜₐₙdₐᵣd - ΔAbₗₐₙₖ ) × D
Parameter Explanation:
Cstd: Creatinine standard concentration, 0.05 mg/mL = 442 μmol/L = 442 nmol/mL
Mr: Molecular weight of creatinine, 113
V₁ : Sample loading volume, 0.006 mL
V₂ : Standard solution loading volume, 0.006 mL
V: Total extraction solution volume, 1 mL
500: Ten thousand cell unit
W: Sample weight (g)
D: Dilution factor; set as 1 without dilution
Cₚᵣ: Sample protein concentration (mg/mL); BCA protein assay kit is recommended for determination.
